17-aryl linker derivatised estrogen 3-sulphamates as inhibitors of steroid sulphatase

ABSTRACT

There is provided a compound comprising a steroidal ring system and a group R 1  selected from any one of a sulphamate group, a phosphonate group, a thiophosphonate group, a sulphonate group or a sulphonamide group; wherein the D ring of the steroidal ring system is substituted by a group R 2  of the formula —L-R 3 , wherein L is an optional linker group and R 3  is an aromatic hydrocarbyl group.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International applicationPCT/GB01/03692 filed Aug. 17, 2001 designating the U.S. and published asWO 02/16393 on Feb. 28, 2002, which claims priority from UK application0020498.2 filed Aug. 18, 2000.

Each of these applications and each of the documents cited in each ofthese applications (“application cited documents”), and each documentreferenced or cited in the application cited documents, either in thetext or during the prosecution of those applications, as well as allarguments in support of patentability advanced during such prosecution,are hereby incorporated herein by reference. Various documents are alsocited in this text (“application cited documents”). Each of theapplication cited documents, and each document cited or referenced inthe application cited documents, is hereby incorporated herein byreference.

FIELD OF INVENTION

The present invention relates to a compound. In particular the presentinvention relates to a compound and to a pharmaceutical compositioncomprising the compound. The present invention also relates to the useof the compound or composition in therapy applications.

BACKGROUND TO THE INVENTION

Evidence suggests that oestrogens are the major mitogens involved inpromoting the growth of tumours in endocrine-dependent tissues, such asthe breast and endometrium. Although plasma oestrogen concentrations aresimilar in women with or without breast cancer, breast tumour oestroneand oestradiol levels are significantly higher than in normal breasttissue or blood. In situ synthesis of oestrogen is thought to make animportant contribution to the high levels of oestrogens in tumours andtherefore inhibitors, in particular specific inhibitors, of oestrogenbiosynthesis are of potential value for the treatment ofendocrine-dependent tumours.

Over the past two decades, there has been considerable interest in thedevelopment of inhibitors of the aromatase pathway—which converts theandrogen precursor androstenedione to oestrone. However, there is nowevidence that the oestrone sulphatase (E1-STS) pathway, i.e. thehydrolysis of oestrone sulphate to oestrone (E1S to E1), as opposed tothe aromatase pathway, is the major source of oestrogen in breasttumours. This theory is supported by a modest reduction of plasmaoestrogen concentration in postmenopausal women with breast cancertreated by aromatase inhibitors, such as aminoglutethimide and4-hydroxyandrostenedione, and also by the fact that plasma E1Sconcentration in these aromatase inhibitor-treated patients remainsrelatively high. The long half-life of E1S in blood (10–12 h) comparedwith the unconjugated oestrogens (20 min) and high levels of steroidsulphatase activity in liver and, normal and malignant breast tissues,also lend support to this theory.

PCT/GB92/01587 teaches novel steroid sulphatase inhibitors andpharmaceutical compositions containing them for use in the treatment ofoestrone dependent tumours, especially breast cancer. These steroidsulphatase inhibitors are sulphamate esters, such as N,N-dimethyloestrone-3-sulphamate and, preferably, oestrone-3-sulphamate (otherwiseknown as “EMATE”). EMATE has the following structure:

It is known that EMATE is a potent E1-STS inhibitor as it displays morethan 99% inhibition of E1-STS activity in intact MCF-7 cells at 0.1 mM.EMATE also inhibits the E1-STS enzyme in a time- andconcentration-dependent manner, indicating that it acts as an activesite-directed inactivator. Although EMATE was originally designed forthe inhibition of E1-STS, it also inhibits dehydroepiandrosteronesulphatase (DHA-STS), which is an enzyme that is believed to have apivotal role in regulating the biosynthesis of the oestrogenic steroidandrostenediol. Also, there is now evidence to suggest thatandrostenediol may be of even greater importance as a promoter of breasttumour growth. EMATE is also active in vivo as almost completeinhibition of rat liver E1-STS (99%) and DHA-STS (99%) activitiesresulted when it is administered either orally or subcutaneously. Inaddition, EMATE has been shown to have a memory enhancing effect inrats. Studies in mice have suggested an association between DHA-STSactivity and the regulation of part of the immune response. It isthought that this may also occur in humans. The bridging O-atom of thesulphamate moiety in EMATE is important for inhibitory activity. Thus,when the 3-O-atom is replaced by other heteroatoms as inoestrone-3-N-sulphamate and oestrone-3-S-sulphamate, these analogues areweaker non-time-dependent inactivators.

Although optimal potency for inhibition of E1-STS may have been attainedin EMATE, it is possible that oestrone may be released during sulphataseinhibition and that EMATE and its oestradiol congener may possessoestrogenic activity.

Ahmed et al (Biochem Biophys Res Commun 1999 Jan. 27; 254(3):811–5)report on a structure-activity relationship study of steroidal andnonsteroidal inhibitors of STS.

The present invention seeks to provide novel compounds suitable for theinhibition of E1-STS as well as other therapeutic applications.

SUMMARY ASPECTS OF THE PRESENT INVENTION

The present invention is based on the surprising finding that certaincompounds could be used as effective steroid sulphatase inhibitorsand/or as agents that can influence cell cycling and/or as agents thatcan influence apoptosis.

In one aspect, the present invention is based on the surprising findingthat certain D ring substituted steroidal compounds could be used aseffective steroid sulphatase inhibitors and/or as modulators of cellcycling and/or as modulators of apoptosis.

The steroidal ring system compounds also includes one or more of asulphamate group, a phosphonate group, a thiophosphonate group, asulphonate group or a sulphonamide group as further substituent(s) onthe ring system. At least one of the sulphamate group, the phosphonategroup, the thiophosphonate group, the sulphonate group or thesulphonamide group is a substituent on the ring component.

The compounds of the present invention may comprise other substituents.These other substituents may, for example, further increase the activityof the compounds of the present invention and/or increase stability (exvivo and/or in vivo).

DETAILED ASPECTS OF THE PRESENT INVENTION

According to one aspect of the present invention, there is provided acompound comprising a steroidal ring system and a group R¹ selected fromany one of a sulphamate group, a phosphonate group, a thiophosphonategroup, a sulphonate group or a sulphonamide group; wherein the D ring ofthe steroidal ring system is substituted by a group R² of the formula-L-R³, wherein L is an optional linker group and R³ is an aromatichydrocarbyl group.

For some compounds of the present invention, it is highly preferred thatthe 17 position of the D ring of the steroidal ring is substituted bygroup R².

According to one aspect of the present invention, there is provided amethod comprising (a) performing a steroid sulphatase assay with one ormore candidate compounds of the present invention; (b) determiningwhether one or more of said candidate compounds is/are capable ofmodulating STS activity and/or cell cycling and/or cell growth and/orapoptosis; and (c) selecting one or more of said candidate compoundsthat is/are capable of modulating STS activity and/or cell cyclingand/or cell growth and/or apoptosis.

According to one aspect of the present invention, there is provided amethod comprising (a) performing a steroid sulphatase assay with one ormore candidate compounds of the present invention; (b) determiningwhether one or more of said candidate compounds is/are capable ofinhibiting STS activity; and (c) selecting one or more of said candidatecompounds that is/are capable of inhibiting STS activity and/or cellcycling and/or cell growth and/or apoptosis.

In any one of the methods of the present invention, one or moreadditional steps may be present. For example, the method may alsoinclude the step of modifying the identified candidate compound (such asby chemical and/or enzymatic techniques) and the optional additionalstep of testing that modified compound for STS inhibition effects (whichmay be to see if the effect is greater or different). By way of furtherexample, the method may also include the step of determining thestructure (such as by use of crystallographic techniques) of theidentified candidate compound and then performing computer modellingstudies—such as to further increase its STS inhibitory action. Thus, thepresent invention also encompasses a computer having a dataset (such asthe crystallographic co-ordinates) for said identified candidatecompound. The present invention also encompasses that identifiedcandidate compound when presented on a computer screen for the analysisthereof—such as protein binding studies.

According to one aspect of the present invention, there is provided acompound identified by the method of the present invention.

According to one aspect of the present invention, there is provided acompound according to the present invention for use in medicine.

According to one aspect of the present invention, there is provided apharmaceutical composition comprising the compound according to thepresent invention optionally admixed with a pharmaceutically acceptablecarrier, diluent, excipient or adjuvant.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for use in the therapy of a condition or diseaseassociated with STS and/or cell cycling and/or apoptosis and/or cellgrowth.

According to one aspect of the present invention, there is provided theuse of a compound according to the present invention in the manufactureof a medicament for use in the therapy of a condition or diseaseassociated with adverse STS levels and/or cell cycling and/or apoptosisand/or cell growth.

The present invention also encompasses the novel compounds of thepresent invention (such as those presented herein), as well as processesfor making same (such as the processes presented herein) as well asnovel intermediates (such as those presented herein) for use in thoseprocesses.

For ease of reference, these and further aspects of the presentinvention are now discussed under appropriate section headings. However,the teachings under each section are not necessarily limited to eachparticular section.

PREFERABLE ASPECTS

Preferably the compound of the present invention is capable ofinhibiting steroid sulphatase (STS) activity and/or is capable of actingas a modulator of cell cycling and/or as a modulator of apoptosis and/oras a modulator of cell growth.

Preferably the compound of the present invention of Formula I

Preferably the compound of the present invention of Formula II

Preferably the compound of the present invention of Formula IIa

Preferably the compound of the present invention of Formula III

Preferably the compound of the present invention of Formula IV

Preferably the compound of the present invention of Formula IVa

Preferably the A ring of the steroidal ring system is substituted with agroup R⁴, wherein R⁴ is a hydrocarbyl group.

In one preferred aspect R⁴ is an oxyhydrocarbyl group.

Preferably R⁴ is an alkoxy group such as C1–10 alkoxy, C1–5 alkoxy, C1,C2 or C3 alkoxy. More preferably the alkoxy group is methoxy.

In one preferred aspect R⁴ is an hydrocarbon group.

Preferably R⁴ is an alkyl group, such as C1–10 alkyl, C1–5 alkyl, C1, C2or C3 alkyl More preferably the alkyl group is ethyl.

In one preferred aspect R⁴ is a group of the formula -L⁴-S-R^(4′),wherein L⁴ is an optional hydrocarbyl linker group, S is sulphur andR^(4′) is a hydrocarbyl group.

Preferably L⁴ and R^(4′) are independently selected from C₁–C₁₀hydrocarbyl, C₁–C₅ hydrocarbyl or C₁–C₃ hydrocarbyl. Preferably L⁴ andR^(4′) are independently selected from hydrocarbon groups, preferablyC₁–C₁₀ hydrocarbon, C₁–C₅ hydrocarbon or C₁–C₃ hydrocarbon. PreferablyL⁴ and R^(4′) are independently selected from alkyl groups, C₁–C₁₀alkyl, C₁–C₅ alkyl or C₁–C₃ alkyl. The hydrocarbyl/hydrocarbon/alkyl ofmay be straight chain or branched and/or may be saturated orunsaturated. In a highly preferred aspect R^(4′) is selected from methyl(—CH₃) and ethyl (—CH₃CH₃).

In one preferred aspect the A ring is substituted with R¹ and R⁴ isortho substituted with respect to R¹.

In one preferred aspect R⁴ is at position 2 of the A ring.

In one preferred aspect the compound of the present invention comprisesat least two or more of sulphamate group, a phosphonate group, athiophosphonate group, a sulphonate group or a sulphonamide group.

Preferably R¹ is a sulphamate group.

In one preferred aspect the compound comprises at least two sulphamategroups. In this aspect preferably the said sulphamate groups are not onthe same ring.

In a preferred aspect the A ring of the present steroidal ring systemcomprises at least one sulphamate group and the D ring of the presentsteroidal ring system comprises at least one sulphamate group.

R³ is an aromatic hydrocarbyl group. The term “aromatic hydrocarbylgroup” used herein means any hydrocarbyl group which contains or formpart of a ring system containing delocalised π electrons.

Preferably R³ is or comprises an aromatic ring. Preferably R³ is anoptionally substituted aromatic ring

Preferably R³ is or comprises a aromatic ring containing carbon andoptionally nitrogen. Preferably R³ is an optionally substituted aromaticring containing carbon and optionally nitrogen.

Preferably R³ is or comprises a six membered aromatic ring. PreferablyR³ is an optionally substituted six membered aromatic ring.

Preferably R³ is or comprises a six membered aromatic ring containingcarbon and optionally nitrogen. Preferably R³ is an optionallysubstituted six membered aromatic ring containing carbon and optionallynitrogen.

Preferably the aromatic ring is substituted with a group R⁵ selectedfrom hydrocarbyl and halogens. Preferably R⁵ is selected fromhydrocarbon and halogens. More preferably R⁵ is selected from alkyl andhalogens. More preferably R⁵ is selected from C₁₋₁₀ alkyl (preferablyC₁₋₅ alkyl), and halogens. More preferably

The linker group L in a preferred aspect is a hydrocarbyl group, morepreferably a hydrocarbon group.

Preferably linker group L is an alkyl group, such as a C₁₋₁₀ alkyl,preferably C₁₋₅ alkyl, preferably C₁ or C₂ alkyl.

The preferred combinations of L and R³ provide preferred groups R² isselected from

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably the compound of the present invention is of the formula

Preferably group R² is in an α conformation.

Preferably group R² is in an α conformation on the 17 position of the Dring.

Preferably the group K and the ring X together will contain, inclusiveof all substituents, a maximum of about 50 carbon atoms, more usually nomore than about 30 to 40 carbon atoms.

For some applications, preferably the compounds have no, or a minimal,oestrogenic effect.

For some applications, preferably the compounds have an oestrogeniceffect.

For some applications, preferably the compounds have a reversibleaction.

For some applications, preferably the compounds have an irreversibleaction.

In one embodiment, the compounds of the present invention are useful forthe treatment of breast cancer.

The present invention also covers novel intermediates that are useful toprepare the compounds of the present invention. For example, the presentinvention covers novel alcohol precursors for the compounds. By way offurther example, the present invention covers bis protected precursorsfor the compounds. Examples of each of these precursors are presentedherein. The present invention also encompasses a process comprising eachor both of those precursors for the synthesis of the compounds of thepresent invention.

SOME ADVANTAGES

One key advantage of the present invention is that the sulphamatecompounds of the present invention can act as STS inhibitors.

Another advantage of the compounds of the present invention is that theymay be potent in vivo.

Some of the compounds of the present invention may be non-oestrogeniccompounds. Here, the term “non-oestrogenic” means exhibiting no orsubstantially no oestrogenic activity.

Another advantage is that some of the compounds may not be capable ofbeing metabolised to compounds which display or induce hormonalactivity.

Some of the compounds of the present invention are also advantageous inthat they may be orally active.

Some of the compounds of the present invention may useful for thetreatment of cancer, such as breast cancer, as well as (or in thealternative) non-malignant conditions, such as the prevention ofauto-immune diseases, particularly when pharmaceuticals may need to beadministered from an early age.

Thus, some of the compounds of the present invention are also believedto have therapeutic uses other than for the treatment ofendocrine-dependent cancers, such as the treatment of autoimmunediseases.

The compounds of the present invention may also be useful as an inducerof apoptosis.

The compounds of the present invention may also be useful as a cellgrowth inhibitors.

STEROID SULPHATASE

Steroid sulphatase—which is sometimes referred to as steroid sulfataseor steryl sulphatase or “STS” for short—hydrolyses several sulphatedsteroids, such as oestrone sulphate, dehydroepiandrosterone sulphate andcholesterol sulphate. STS has been allocated the enzyme number EC3.1.6.2.

STS has been cloned and expressed. For example see Stein et al (J. Biol.Chem. 264:13865–13872 (1989)) and Yen et al (Cell 49:443–454(1987)).

STS is an enzyme that has been implicated in a number of diseaseconditions.

By way of example, workers have found that a total deficiency in STSproduces ichthyosis. According to some workers, STS deficiency is fairlyprevalent in Japan. The same workers (Sakura et al, J Inherit Metab Dis1999 November; 20(6):807–10) have also reported that allergicdiseases—such as bronchial asthma, allergic rhinitis, or atopicdermatitis—may be associated with a steroid sulphatase deficiency.

In addition to disease states being brought on through a total lack ofSTS activity, an increased level of STS activity may also bring aboutdisease conditions. By way of example, and as indicated above, there isstrong evidence to support a role of STS in breast cancer growth andmetastasis.

STS has also been implicated in other disease conditions. By way ofexample, Le Roy et al (Behav Genet 1999 March; 29(2):131–6) havedetermined that there may be a genetic correlation between steroidsulphatase concentration and initiation of attack behaviour in mice. Theauthors conclude that sulphatation of steroids may be the prime mover ofa complex network, including genes shown to be implicated in aggressionby mutagenesis.

STS INHIBITION

It is believed that some disease conditions associated with STS activityare due to conversion of a nonactive, sulphated oestrone to an active,nonsulphated oestrone. In disease conditions associated with STSactivity, it would be desirable to inhibit STS activity.

Here, the term “inhibit” includes reduce and/or eliminate and/or maskand/or prevent the detrimental action of STS.

STS INHIBITOR

In accordance with the present invention, the compound of the presentinvention is capable of acting as an STS inhibitor.

Here, the term “inhibitor” as used herein with respect to the compoundof the present invention means a compound that can inhibit STSactivity—such as reduce and/or eliminate and/or mask and/or prevent thedetrimental action of STS. The STS inhibitor may act as an antagonist.

The ability of compounds to inhibit oestrone sulphatase activity can beassessed using either intact MCF-7 breast cancer cells or placentalmicrosomes. In addition, an animal model may be used. Details onsuitable Assay Protocols are presented in following sections. It is tobe noted that other assays could be used to determine STS activity andthus STS inhibition. For example, reference may also be made to theteachings of WO-A-99/50453.

Preferably, for some applications, the compound is further characterisedby the feature that if the sulphamate group were to be substituted by asulphate group to form a sulphate derivative, then the sulphatederivative would be hydrolysable by an enzyme having steroid sulphatase(E.C. 3.1.6.2) activity—i.e. when incubated with steroid sulphatase EC3.1.6.2 at pH 7.4 and 37° C.

In one preferred embodiment, if the sulphamate group of the compoundwere to be replaced with a sulphate group to form a sulphate compoundthen that sulphate compound would be hydrolysable by an enzyme havingsteroid sulphatase (E.C. 3.1.6.2) activity and would yield a Km value ofless than 200 mmolar, preferably less than 150 mmolar, preferably lessthan 100 mmolar, preferably less than 75 mmolar, preferably less than 50mmolar, when incubated with steroid sulphatase EC 3.1.6.2 at pH 7.4 and37° C.

In a preferred embodiment, the compound of the present invention is nothydrolysable by an enzyme having steroid sulphatase (E.C. 3.1.6.2)activity.

For some applications, preferably the compound of the present inventionhas at least about a 100 fold selectivity to a desired target (e.g.STS), preferably at least about a 150 fold selectivity to the desiredtarget, preferably at least about a 200 fold selectivity to the desiredtarget, preferably at least about a 250 fold selectivity to the desiredtarget, preferably at least about a 300 fold selectivity to the desiredtarget, preferably at least about a 350 fold selectivity to the desiredtarget.

It is to be noted that the compound of the present invention may haveother beneficial properties in addition to or in the alternative to itsability to inhibit STS activity.

HYDROCARBYL

The term “hydrocarbyl group” as used herein means a group comprising atleast C and H and may optionally comprise one or more other suitablesubstituents. Examples of such substituents may include halo, alkoxy,nitro, an alkyl group, a cyclic group etc. In addition to thepossibility of the substituents being a cyclic group, a combination ofsubstituents may form a cyclic group. If the hydrocarbyl group comprisesmore than one C then those carbons need not necessarily be linked toeach other. For example, at least two of the carbons may be linked via asuitable element or group. Thus, the hydrocarbyl group may containhetero atoms. Suitable hetero atoms will be apparent to those skilled inthe art and include, for instance, sulphur, nitrogen and oxygen. Anon-limiting example of a hydrocarbyl group is an acyl group.

A typical hydrocarbyl group is a hydrocarbon group. Here the term“hydrocarbon” means any one of an alkyl group, an alkenyl group, analkynyl group, which groups may be linear, branched or cyclic, or anaryl group. The term hydrocarbon also includes those groups but whereinthey have been optionally substituted. If the hydrocarbon is a branchedstructure having substituent(s) thereon, then the substitution may be oneither the hydrocarbon backbone or on the branch; alternatively thesubstitutions may be on the hydrocarbon backbone and on the branch.

SULPHAMATE GROUP

In one embodiment, the ring X has a sulphamate group as a substituent.The term “sulphamate” as used herein includes an ester of sulphamicacid, or an ester of an N-substituted derivative of sulphamic acid, or asalt thereof.

If R³ is a sulphamate group then the compound of the present inventionis referred to as a sulphamate compound.

Typically, the sulphamate group has the formula:(R⁴)(R⁵)N—S(O)(O)—O—wherein preferably R⁴ and R⁵ are independently selected from H, alkyl,cycloalkyl, alkenyl and aryl, or combinations thereof, or togetherrepresent alkylene, wherein the or each alkyl or cycloalkyl or alkenylor optionally contain one or more hetero atoms or groups.

When substituted, the N-substituted compounds of this invention maycontain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-arylsubstituents, preferably containing or each containing a maximum of 10carbon atoms. When R⁴ and/or R⁵ is alkyl, the preferred values are thosewhere R⁴ and R⁵ are each independently selected from lower alkyl groupscontaining from 1 to 6 carbon atoms, that is to say methyl, ethyl,propyl etc. R⁴ and R⁵ may both be methyl. When R⁴ and/or R⁵ is aryl,typical values are phenyl and tolyl (PhCH₃; o). Where R⁴ and R⁵represent cycloalkyl, typical values are cyclopropyl, cyclopentyl,cyclohexyl etc. When joined together R⁴ and R⁵ typically represent analkylene group providing a chain of 4 to 6 carbon atoms, optionallyinterrupted by one or more hetero atoms or groups, e.g. to provide a 5membered heterocycle, e.g. morpholino, pyrrolidino or piperidino.

Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groupsare included containing as substituents therein one or more groups whichdo not interfere with the sulphatase inhibitory activity of the compoundin question. Exemplary non-interfering substituents include hydroxy,amino, halo, alkoxy, alkyl and aryl.

In some embodiments, the sulphamate group may form a ring structure bybeing fused to (or associated with) one or more atoms in or on group X.

In some embodiments, there may be more than one sulphamate group. By wayof example, there may be two sulphamates (i.e. bis-sulphamatecompounds). If these compounds are based on a steroidal nucleus,preferably the second (or at least one of the additional) sulphamategroup is located at position 17 of the steroidal nucleus. These groupsneed not be the same.

In some preferred embodiments, at least one of R⁴ and R⁵ is H.

In some further preferred embodiments, each of R⁴ and R⁵ is H.

PHOSPHONATE GROUP

If R³ is a phosphonate group then the compound of the present inventionis referred to as a phosphonate compound.

Typically, the phosphonate group has the formula:(R⁶)—P(O)(OH)—O—wherein preferably R⁶ is H, alkyl, cycloalkyl, alkenyl or aryl, orcombinations thereof, wherein the or each alkyl or cycloalkyl or alkenylor optionally contain one or more hetero atoms or groups.

When substituted, the N-substituted compounds of this invention maycontain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-arylsubstituents, preferably containing or each containing a maximum of 10carbon atoms. When R⁶ is alkyl, R⁶ may be a lower alkyl groupscontaining from 1 to 6 carbon atoms, that is to say methyl, ethyl,propyl etc. By way of example, R⁶ may be methyl. When R⁶ is aryl,typical values are phenyl and tolyl (PhCH₃;o). Where R⁶ representscycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.R⁶ may even comprise an alkylene group providing a chain of 4 to 6carbon atoms, optionally interrupted by one or more hetero atoms orgroups, e.g. to provide a 5 membered heterocycle, e.g. morpholino,pyrrolidino or piperidino.

Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groupsare included containing as substituents therein one or more groups whichdo not interfere with the sulphatase inhibitory activity of the compoundin question. Exemplary non-interfering substituents include hydroxy,amino, halo, alkoxy, alkyl and aryl.

In some embodiments, the phosphonate group may form a ring structure bybeing fused to (or associated with) one or more atoms in or on group X.

In some embodiments, there may be more than one phosphonate group. Byway of example, there may be two phosphonates (i.e. bis-phosphonatecompounds). If these compounds are based on a steroidal nucleus,preferably the second (or at least one of the additional) phosphonategroup is located at position 17 of the steroidal nucleus. These groupsneed not be the same.

THIOPHOSPHONATE GROUP

If R³ is a thiophosphonate group then the compound of the presentinvention is referred to as a thiophosphonate compound.

Typically, the thiophosphonate group has the formula:(R⁷)—P(S)(OH)—O—wherein preferably R⁷ is H, alkyl, cycloalkyl, alkenyl or aryl, orcombinations thereof, wherein the or each alkyl or cycloalkyl or alkenylor optionally contain one or more hetero atoms or groups.

When substituted, the N-substituted compounds of this invention maycontain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-arylsubstituents, preferably containing or each containing a maximum of 10carbon atoms. When R⁷ is alkyl, R⁷ may be a lower alkyl groupscontaining from 1 to 6 carbon atoms, that is to say methyl, ethyl,propyl etc. By way of example, R⁷ may be methyl. When R⁷ is aryl,typical values are phenyl and tolyl (PhCH₃;o). Where R⁷ representscycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.R⁷ may even comprise an alkylene group providing a chain of 4 to 6carbon atoms, optionally interrupted by one or more hetero atoms orgroups, e.g. to provide a 5 membered heterocycle, e.g. morpholino,pyrrolidino or piperidino.

Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groupsare included containing as substituents therein one or more groups whichdo not interfere with the sulphatase inhibitory activity of the compoundin question. Exemplary non-interfering substituents include hydroxy,amino, halo, alkoxy, alkyl and aryl.

In some embodiments, the thiophosphonate group may form a ring structureby being fused to (or associated with) one or more atoms in or on groupX.

In some embodiments, there may be more than one thiophosphonate group.By way of example, there may be two thiophosphonates (i.e.bis-thiophosphonate compounds). If these compounds are based on asteroidal nucleus, preferably the second (or at least one of theadditional) thiophosphonate group is located at position 17 of thesteroidal nucleus. These groups need not be the same.

SULPHONATE GROUP

If R³ is a sulphonate group then the compound of the present inventionis referred to as a sulphonate compound.

Typically, the sulphonate group has the formula:(R⁸)—S(O)(O)—O—wherein preferably R⁸ is H, alkyl, cycloalkyl, alkenyl or aryl, orcombinations thereof, wherein the or each alkyl or cycloalkyl or alkenylor optionally contain one or more hetero atoms or groups.

When substituted, the N-substituted compounds of this invention maycontain one or two N-alkyl, N-alkenyl, N-cycloalkyl or N-arylsubstituents, preferably containing or each containing a maximum of 10carbon atoms. When R⁸ is alkyl, R⁸ may be a lower alkyl groupscontaining from 1 to 6 carbon atoms, that is to say methyl, ethyl,propyl etc. By way of example, R⁸ may be methyl. When R⁸ is aryl,typical values are phenyl and tolyl (PhCH₃;o). Where R⁸ representscycloalkyl, typical values are cyclopropyl, cyclopentyl, cyclohexyl etc.R⁸ may even comprise an alkylene group providing a chain of 4 to 6carbon atoms, optionally interrupted by one or more hetero atoms orgroups, e.g. to provide a 5 membered heterocycle, e.g. morpholino,pyrrolidino or piperidino.

Within the values alkyl, cycloalkyl, alkenyl and aryl substituted groupsare included containing as substituents therein one or more groups whichdo not interfere with the sulphatase inhibitory activity of the compoundin question. Exemplary non-interfering substituents include hydroxy,amino, halo, alkoxy, alkyl and aryl.

In some embodiments, the sulphonate group may form a ring structure bybeing fused to (or associated with) one or more atoms in or on group X.

In some embodiments, there may be more than one sulphonate group. By wayof example, there may be two sulphonates (i.e. bis-sulphonatecompounds). If these compounds are based on a steroidal nucleus,preferably the second (or at least one of the additional) sulphonategroup is located at position 17 of the steroidal nucleus. These groupsneed not be the same.

COMBINATION OF SULPHONATE/PHOSPHONATE/THIOPHOSPHONATE/SULPHAMATE

For some compounds of the present invention there may be present one ofa sulphonate as herein defined or a phosphonate as herein defined or athiophosphonate as herein defined or a sulphamate as herein defined; andanother of a sulphonate as herein defined or a phosphonate as hereindefined or a thiophosphonate as herein defined or a sulphamate as hereindefined. By way of example, the compound of the present invention maycomprise one sulphamate group and one phosphonate group.

If these compounds of the present invention are based on a steroidalnucleus, preferably the other of said groups is located at position 17of the steroidal nucleus.

MIMIC

In one aspect, the “steroidal ring system” can be a mimic of a steroidalring structure

The term “mimic” as used herein means having a similar or differentstructure but having a similar functional effect. In other words, thesteroidal ring system may be a bio-isostere of the rings of a steroid,or an active part thereof.

In a preferred aspect, the steroidal ring system may be a bio-isostereof the rings of oestrone, or a part thereof.

STEROIDAL RING STRUCTURE

As it is well known in the art, a classical steroidal ring structure hasthe generic formula of:

In the above formula, the rings have been labelled in the conventionalmanner.

An example of a bio-isostere is when any one or more of rings A, B, Cand D is a heterocyclic ring and/or when any one or more of rings A, B,C and D has been substituted and/or when any one or more of rings A, B,C and D has been modified; but wherein the bio-isostere in the absenceof the sulphamate group has steroidal properties.

In this regard, the structure of a preferred polycyclic structure can bepresented as:

wherein each ring A′, B′, C′ and D′ independently represents aheterocyclic ring or a non-heterocyclic ring, which rings may beindependently substituted or unsubstituted, saturated or unsaturated.

By way of example, any one or more of rings A′, B′, C′ and D′ may beindependently substituted with suitable groups—such as an alkyl group,an aryl group, a hydroxy group, a halo group, a hydrocarbyl group, anoxyhydrocarbyl group etc.

An example of D′ is a five or six membered non-heterocyclic ring havingat least one substituent.

In one preferred embodiment, the ring D′ is substituted with a ethinylgroup.

If any one of rings A′, B′, C′ and D′ is a heterocyclic ring, thenpreferably that heterocyclic ring comprises a combination of C atoms andat least one N atom and/or at least one O atom. Other heterocyclic atomsmay be present in the ring.

Examples of suitable, preferred steroidal nuclei rings A′–D′ of thecompounds of the present invention include rings A–D ofdehydroepiandrosterone and oestrogens including oestrone.

Preferred steroidal nuclei rings A′-D′ of the compounds of the presentinvention include rings A–D of:

-   oestrones and substituted oestrones, viz:    -   oestrone    -   2-OH-oestrone    -   4-OH-oestrone    -   6α-OH-oestrone    -   7α-OH-oestrone    -   16α-OH-oestrone    -   16β-OH-oestrone    -   2-MeO-oestrone    -   17-deoxyoestrone-   oestradiols and substituted oestradiols, viz:    -   4-OH-17β-oestradiol    -   6α-OH-17β-oestradiol    -   7α-OH-17β-oestradiol    -   4-OH-17α-oestradiol    -   6α-OH-17α-oestradiol    -   7α-OH-17α-oestradiol    -   16α-OH-17α-oestradiol    -   16α-OH-17β-oestradiol    -   16β-OH-17α-oestradiol    -   16β-OH-17β-oestradiol    -   17α-oestradiol    -   17β-oestradiol    -   17α-ethinyl-17β-oestradiol    -   17β-ethinyl-17α-oestradiol    -   17-deoxyoestradiol-   oestriols and substituted oestriols, viz:    -   oestriol    -   4-OH-oestriol    -   6α-OH-oestriol    -   7α-OH-oestriol    -   17-deoxyoestriol-   dehydroepiandrosterones and substituted dehydroepiandrosterones,    viz:    -   dehydroepiandrosterones    -   6α-OH-dehydroepiandrosterone    -   7α-OH-dehydroepiandrosterone    -   16α-OH-dehydroepiandrosterone    -   16β-OH-dehydroepiandrosterone    -   androstenediol

In general terms the ring system A′B′C′D′ may contain a variety ofnon-interfering substituents. In particular, the ring system A′B′C′D′may contain one or more hydroxy, alkyl especially lower (C₁–C₆) alkyl,e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl,n-pentyl and other pentyl isomers, and n-hexyl and other hexyl isomers,alkoxy especially lower (C₁–C₆) alkoxy, e.g. methoxy, ethoxy, propoxyetc., alkinyl, e.g. ethinyl, or halogen, e.g. fluoro substituents.

NON-STEROID STRUCTURES

In an alternative embodiment, the compound of the present invention maynot contain or be based on a steroid nucleus. In this regard, thepolycyclic compound may contain or be based on a non-steroidal ringsystem—such as diethylstilboestrol, stilboestrol, coumarins, flavonoids,combrestatin and other ring systems. Other suitable non-steroidalcompounds for use in or as the composition of the present invention maybe found in U.S. Pat. No. 5,567,831.

OTHER SUBSTITUENTS

The compound of the present invention may have substituents other thanR¹ and R². By way of example, these other substituents may be one ormore of: one or more sulphamate group(s), one or more phosphonategroup(s), one or more thiophosphonate group(s), one or more sulphonategroup(s), one or more sulphonamide group(s), one or more halo groups,one or more O groups, one or more hydroxy groups, one or more aminogroups, one or more sulphur containing group(s), one or more hydrocarbylgroup(s)—such as an oxyhydrocarbyl group.

OXYHYDROCARBYL

The term “oxyhydrocarbyl” group as used herein means a group comprisingat least C, H and O and may optionally comprise one or more othersuitable substituents. Examples of such substituents may include halo-,alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to thepossibility of the substituents being a cyclic group, a combination ofsubstituents may form a cyclic group. If the oxyhydrocarbyl groupcomprises more than one C then those carbons need not necessarily belinked to each other. For example, at least two of the carbons may belinked via a suitable element or group. Thus, the oxyhydrocarbyl groupmay contain hetero atoms. Suitable hetero atoms will be apparent tothose skilled in the art and include, for instance, sulphur andnitrogen.

In one embodiment of the present invention, the oxyhydrocarbyl group isa oxyhydrocarbon group.

Here the term “oxyhydrocarbon” means any one of an alkoxy group, anoxyalkenyl group, an oxyalkynyl group, which groups may be linear,branched or cyclic, or an oxyaryl group. The term oxyhydrocarbon alsoincludes those groups but wherein they have been optionally substituted.If the oxyhydrocarbon is a branched structure having substituent(s)thereon, then the substitution may be on either the hydrocarbon backboneor on the branch; alternatively the substitutions may be on thehydrocarbon backbone and on the branch.

Typically, the oxyhydrocarbyl group is of the formula C₁₋₆O (such as aC₁₋₃O).

ASSAY FOR DETERMINING STS ACTIVITY USING CANCER CELLS (PROTOCOL 1)

Inhibition of Steroid Sulphatase Activity in MCF-7 Cells

Steroid sulphatase activity is measured in vitro using intact MCF-7human breast cancer cells. This hormone dependent cell line is widelyused to study the control of human breast cancer cell growth. Itpossesses significant steroid sulphatase activity (MacIndoe et al.Endocrinology, 123, 1281–1287 (1988); Purohit & Reed, Int. J. Cancer,50, 901–905 (1992)) and is available in the U.S.A. from the AmericanType Culture Collection (ATCC) and in the U.K. (e.g. from The ImperialCancer Research Fund).

Cells are maintained in Minimal Essential Medium (MEM) (FlowLaboratories, Irvine, Scotland) containing 20 mM HEPES, 5% foetal bovineserum, 2 mM glutamine, non-essential amino acids and 0.075% sodiumbicarbonate. Up to 30 replicate 25 cm2 tissue culture flasks are seededwith approximately 1×10⁵ cells/flask using the above medium. Cells aregrown to 80% confluency and the medium is changed every third day.

Intact monolayers of MCF-7 cells in triplicate 25 cm² tissue cultureflasks are washed with Earle's Balanced Salt Solution (EBSS from ICNFlow, High Wycombe, U.K.) and incubated for 3–4 hours at 37° C. with 5pmol (7×10⁵ dpm) [6,7–3H]oestrone-3-sulphate (specific activity 60Ci/mmol from New England Nuclear, Boston, Mass., U.S.A.) in serum-freeMEM (2.5 ml) together with oestrone-3-sulphamate (11 concentrations: 0;1 fM; 0.01 pM; 0.1 pM; 1 pM; 0.01 nM; 0.1 nM; 1 nM; 0.01 mM; 0.1 mM; 1mM). After incubation each flask is cooled and the medium (1 ml) ispipetted into separate tubes containing [14C]oestrone (7×103 dpm)(specific activity 97 Ci/mmol from Amersham International RadiochemicalCentre, Amersham, U.K.). The mixture is shaken thoroughly for 30 secondswith toluene (5 ml). Experiments have shown that >90% [14C] oestrone and<0.1% [3H]oestrone-3-sulphate is removed from the aqueous phase by thistreatment. A portion (2 ml) of the organic phase is removed, evaporatedand the 3H and 14C content of the residue determined by scintillationspectrometry. The mass of oestrone-3-sulphate hydrolysed was calculatedfrom the 3H counts obtained (corrected for the volumes of the medium andorganic phase used, and for recovery of [14C] oestrone added) and thespecific activity of the substrate. Each batch of experiments includesincubations of microsomes prepared from a sulphatase-positive humanplacenta (positive control) and flasks without cells (to assess apparentnon-enzymatic hydrolysis of the substrate). The number of cell nucleiper flask is determined using a Coulter Counter after treating the cellmonolayers with Zaponin. One flask in each batch is used to assess cellmembrane status and viability using the Trypan Blue exclusion method(Phillips, H. J. (1973) In: Tissue culture and applications, [eds:Kruse, D. F. & Patterson, M. K.]; pp. 406–408; Academic Press, NewYork).

Results for steroid sulphatase activity are expressed as the mean ±1S.D. of the total product (oestrone+oestradiol) formed during theincubation period (20 hours) calculated for 106 cells and, for valuesshowing statistical significance, as a percentage reduction (inhibition)over incubations containing no oestrone-3-sulphamate. Unpaired Student'st-test was used to test the statistical significance of results.

ASSAY FOR DETERMINING STS ACTIVITY USING PLACENTAL MICROSOMES (PROTOCOL2)

Inhibition of Steroid Sulphatase Activity in Placental Microsomes

Sulphatase-positive human placenta from normal term pregnancies arethoroughly minced with scissors and washed once with cold phosphatebuffer (pH 7.4, 50 mM) then re-suspended in cold phosphate buffer (5ml/g tissue). Homogenisation is accomplished with an Ultra-Turraxhomogeniser, using three 10 second bursts separated by 2 minute coolingperiods in ice. Nuclei and cell debris are removed by centrifuging (4°C.) at 2000 g for 30 minutes and portions (2 ml) of the supernatant arestored at 20° C. The protein concentration of the supernatants isdetermined by the method of Bradford (Anal. Biochem., 72, 248–254(1976)).

Incubations (1 ml) are carried out using a protein concentration of 100mg/ml, substrate concentration of 20 mM [6,7–3H]oestrone-3-sulphate(specific activity 60 Ci/mmol from New England Nuclear, Boston, Mass.,U.S.A.) and an incubation time of 20 minutes at 37° C. If necessaryeight concentrations of compounds are employed: 0 (i.e. control); 0.05mM; 0.1 mM; 0.2 mM; 0.4 mM; 0.6 mM; 0.8 mM; 1.0 mM. After incubationeach sample is cooled and the medium (1 ml) was pipetted into separatetubes containing [14C]oestrone (7×103 dpm) (specific activity 97 Ci/mmolfrom Amersham International Radiochemical Centre, Amersham, U.K.). Themixture is shaken thoroughly for 30 seconds with toluene (5 ml).Experiments have shown that >90% [14C]oestrone and <0.1%[3H]oestrone-3-sulphate is removed from the aqueous phase by thistreatment. A portion (2 ml) of the organic phase was removed, evaporatedand the 3H and 14C content of the residue determined by scintillationspectrometry. The mass of oestrone-3-sulphate hydrolysed is calculatedfrom the 3H counts obtained (corrected for the volumes of the medium andorganic phase used, and for recovery of [14C]oestrone added) and thespecific activity of the substrate.

ANIMAL ASSAY MODEL FOR DETERMINING STS ACTIVITY (PROTOCOL 3)

Inhibition of Oestrone Sulphatase Activity in vivo

The compounds of the present invention may be studied using an animalmodel, in particular in ovariectomised rats. In this model compoundswhich are oestrogenic stimulate uterine growth.

The compound (0.1 mg/Kg/day for five days) is administered orally torats with another group of animals receiving vehicle only (propyleneglycol). At the end of the study samples of liver tissue were obtainedand oestrone sulphatase activity assayed using 3H oestrone sulphate asthe substrate as previously described (see PCT/GB95/02638).

ANIMAL ASSAY MODEL FOR DETERMINING OESTROGENIC ACTIVITY (PROTOCOL 4)

Lack of in vivo Oestrogenicity

The compounds of the present invention may be studied using an animalmodel, in particular in ovariectomised rats. In this model, compoundswhich are oestrogenic stimulate uterine growth.

The compound (0.1 mg/Kg/day for five days) was administered orally torats with another group of animals receiving vehicle only (propyleneglycol). At the end of the study uteri were obtained and weighed withthe results being expressed as uterine weight/whole body weight×100.

Compounds having no significant effect on uterine growth are notoestrogenic.

BIOTECHNOLOGICAL ASSAYS FOR DETERMINING STS ACTIVITY (PROTOCOL 5)

The ability of compounds to inhibit oestrone sulphatase activity canalso be assessed using amino acid sequences or nucleotide sequencesencoding STS, or active fragments, derivatives, homologues or variantsthereof in, for example, high-through put screens.

Any one or more of appropriate targets—such as an amino acid sequenceand/or nucleotide sequence—may be used for identifying an agent capableof modulating STS in any of a variety of drug screening techniques. Thetarget employed in such a test may be free in solution, affixed to asolid support, borne on a cell surface, or located intracellularly. Theabolition of target activity or the formation of binding complexesbetween the target and the agent being tested may be measured.

The assay of the present invention may be a screen, whereby a number ofagents are tested. In one aspect, the assay method of the presentinvention is a high through put screen.

Techniques for drug screening may be based on the method described inGeysen, European Patent Application 84/03564, published on Sep. 13,1984. In summary, large numbers of different small peptide testcompounds are synthesised on a solid substrate, such as plastic pins orsome other surface. The peptide test compounds are reacted with asuitable target or fragment thereof and washed. Bound entities are thendetected—such as by appropriately adapting methods well known in theart. A purified target can also be coated directly onto plates for usein a drug screening techniques. Alternatively, non-neutralisingantibodies can be used to capture the peptide and immobilise it on asolid support.

This invention also contemplates the use of competitive drug screeningassays in which neutralising antibodies capable of binding a targetspecifically compete with a test compound for binding to a target.

Another technique for screening provides for high throughput screening(HTS) of agents having suitable binding affinity to the substances andis based upon the method described in detail in WO 84/03564.

It is expected that the assay methods of the present invention will besuitable for both small and large-scale screening of test compounds aswell as in quantitative assays.

In one preferred aspect, the present invention relates to a method ofidentifying agents that selectively modulate STS, which compounds havethe formula (Ia).

REPORTERS

A wide variety of reporters may be used in the assay methods (as well asscreens) of the present invention with preferred reporters providingconveniently detectable signals (e.g. by spectroscopy). By way ofexample, a reporter gene may encode an enzyme which catalyses a reactionwhich alters light absorption properties.

Other protocols include enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA) and fluorescent activated cell sorting (FACS). Atwo-site, monoclonal-based immunoassay utilising monoclonal antibodiesreactive to two non-interfering epitopes may even be used. These andother assays are described, among other places, in Hampton R et al(1990, Serological Methods, A Laboratory Manual, APS Press, St PaulMinn.) and Maddox DE et al (1983, J Exp Med 15 8:121 1).

Examples of reporter molecules include but are not limited to(β-galactosidase, invertase, green fluorescent protein, luciferase,chloramphenicol, acetyltransferase, (-glucuronidase, exo-glucanase andglucoamylase. Alternatively, radiolabelled or fluorescent tag-labellednucleotides can be incorporated into nascent transcripts which are thenidentified when bound to oligonucleotide probes.

By way of further examples, a number of companies such as PharmaciaBiotech (Piscataway, N.J.), Promega (Madison, Wis.), and US BiochemicalCorp (Cleveland, Ohio) supply commercial kits and protocols for assayprocedures. Suitable reporter molecules or labels include thoseradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particlesand the like. Patents teaching the use of such labels include U.S. Pat.No. 3,817,837; U.S. Pat. No. 3,850,752; U.S. Pat. No. 3,939,350; U.S.Pat. No. 3,996,345; U.S. Pat. No. 4,277,437; U.S. Pat. No. 4,275,149 andU.S. Pat. No. 4,366,241.

HOST CELLS

The term “host cell”—in relation to the present invention includes anycell that could comprise the target for the agent of the presentinvention.

Thus, a further embodiment of the present invention provides host cellstransformed or transfected with a polynucleotide that is or expressesthe target of the present invention. Preferably said polynucleotide iscarried in a vector for the replication and expression ofpolynucleotides that are to be the target or are to express the target.The cells will be chosen to be compatible with the said vector and mayfor example be prokaryotic (for example bacterial), fungal, yeast orplant cells.

The gram negative bacterium E. coli is widely used as a host forheterologous gene expression. However, large amounts of heterologousprotein tend to accumulate inside the cell. Subsequent purification ofthe desired protein from the bulk of E. coli intracellular proteins cansometimes be difficult.

In contrast to E. coli, bacteria from the genus Bacillus are verysuitable as heterologous hosts because of their capability to secreteproteins into the culture medium. Other bacteria suitable as hosts arethose from the genera Streptomyces and Pseudomonas.

Depending on the nature of the polynucleotide encoding the polypeptideof the present invention, and/or the desirability for further processingof the expressed protein, eukaryotic hosts such as yeasts or other fungimay be preferred. In general, yeast cells are preferred over fungalcells because they are easier to manipulate. However, some proteins areeither poorly secreted from the yeast cell, or in some cases are notprocessed properly (e.g. hyperglycosylation in yeast). In theseinstances, a different fungal host organism should be selected.

Examples of suitable expression hosts within the scope of the presentinvention are fungi such as Aspergillus species (such as those describedin EP-A-0184438 and EP-A-0284603) and Trichoderma species; bacteria suchas Bacillus species (such as those described in EP-A-0134048 andEP-A-0253455), Streptomyces species and Pseudomonas species; and yeastssuch as Kluyveromyces species (such as those described in EP-A-0096430and EP-A-0301670) and Saccharomyces species. By way of example, typicalexpression hosts may be selected from Aspergillus niger, Aspergillusniger var. tubigenis, Aspergillus niger var. awamori, Aspergillusaculeatis, Aspergillus nidulans, Aspergillus orvzae, Trichoderma reesei,Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens,Kluyveromyces lactis and Saccharomyces cerevisiae.

The use of suitable host cells—such as yeast, fungal and plant hostcells—may provide for post-translational modifications (e.g.myristoylation, glycosylation, truncation, lapidation and tyrosine,serine or threonine phosphorylation) as may be needed to confer optimalbiological activity on recombinant expression products of the presentinvention.

ORGANISM

The term “organism” in relation to the present invention includes anyorganism that could comprise the target according to the presentinvention and/or products obtained therefrom. Examples of organisms mayinclude a fungus, yeast or a plant.

The term “transgenic organism” in relation to the present inventionincludes any organism that comprises the target according to the presentinvention and/or products obtained.

TRANSFORMATION OF HOST CELLS/HOST ORGANISMS

As indicated earlier, the host organism can be a prokaryotic or aeukaryotic organism. Examples of suitable prokaryotic hosts include E.coli and Bacillus subtilis. Teachings on the transformation ofprokaryotic hosts is well documented in the art, for example seeSambrook et al (Molecular Cloning: A Laboratory Manual, 2nd edition,1989, Cold Spring Harbor Laboratory Press) and Ausubel et al., CurrentProtocols in Molecular Biology (1995), John Wiley & Sons, Inc.

If a prokaryotic host is used then the nucleotide sequence may need tobe suitably modified before transformation—such as by removal ofintrons.

In another embodiment the transgenic organism can be a yeast. In thisregard, yeast have also been widely used as a vehicle for heterologousgene expression. The species Saccharomyces cerevisiae has a long historyof industrial use, including its use for heterologous gene expression.Expression of heterologous genes in Saccharomyces cerevisiae has beenreviewed by Goodey et al (1987, Yeast Biotechnology, D R Berry et al,eds, pp 401–429, Allen and Unwin, London) and by King et al (1989,Molecular and Cell Biology of Yeasts, E F Walton and G T Yarronton, eds,pp 107–133, Blackie, Glasgow).

For several reasons Saccharomyces cerevisiae is well suited forheterologous gene expression. First, it is non-pathogenic to humans andit is incapable of producing certain endotoxins. Second, it has a longhistory of safe use following centuries of commercial exploitation forvarious purposes. This has led to wide public acceptability. Third, theextensive commercial use and research devoted to the organism hasresulted in a wealth of knowledge about the genetics and physiology aswell as large-scale fermentation characteristics of Saccharomycescerevisiae.

A review of the principles of heterologous gene expression inSaccharomyces cerevisiae and secretion of gene products is given by EHinchcliffe E Kenny (1993, “Yeast as a vehicle for the expression ofheterologous genes”, Yeasts, Vol 5, Anthony H Rose and J StuartHarrison, eds, 2nd edition, Academic Press Ltd.).

Several types of yeast vectors are available, including integrativevectors, which require recombination with the host genome for theirmaintenance, and autonomously replicating plasmid vectors.

In order to prepare the transgenic Saccharomyces, expression constructsare prepared by inserting the nucleotide sequence into a constructdesigned for expression in yeast. Several types of constructs used forheterologous expression have been developed. The constructs contain apromoter active in yeast fused to the nucleotide sequence, usually apromoter of yeast origin, such as the GAL1 promoter, is used. Usually asignal sequence of yeast origin, such as the sequence encoding the SUC2signal peptide, is used. A terminator active in yeast ends theexpression system.

For the transformation of yeast several transformation protocols havebeen developed. For example, a transgenic Saccharomyces according to thepresent invention can be prepared by following the teachings of Hinnenet al (1978, Proceedings of the National Academy of Sciences of the USA75, 1929); Beggs, J D (1978, Nature, London, 275, 104); and Ito, H et al(1983, J Bacteriology 153, 163–168).

The transformed yeast cells are selected using various selectivemarkers. Among the markers used for transformation are a number ofauxotrophic markers such as LEU2, HIS4 and TRP1, and dominant antibioticresistance markers such as aminoglycoside antibiotic markers, e.g. G418.

Another host organism is a plant. The basic principle in theconstruction of genetically modified plants is to insert geneticinformation in the plant genome so as to obtain a stable maintenance ofthe inserted genetic material. Several techniques exist for insertingthe genetic information, the two main principles being directintroduction of the genetic information and introduction of the geneticinformation by use of a vector system. A review of the generaltechniques may be found in articles by Potrykus (Annu Rev Plant PhysiolPlant Mol Biol [1991] 42:205–225) and Christou (Agro-Food-IndustryHi-Tech March/April 1994 17–27). Further teachings on planttransformation may be found in EP-A-0449375.

Thus, the present invention also provides a method of transforming ahost cell with a nucleotide sequence that is to be the target or is toexpress the target. Host cells transformed with the nucleotide sequencemay be cultured under conditions suitable for the expression of theencoded protein. The protein produced by a recombinant cell may bedisplayed on the surface of the cell. If desired, and as will beunderstood by those of skill in the art, expression vectors containingcoding sequences can be designed with signal sequences which directsecretion of the coding sequences through a particular prokaryotic oreukaryotic cell membrane. Other recombinant constructions may join thecoding sequence to nucleotide sequence encoding a polypeptide domainwhich will facilitate purification of soluble proteins (Kroll D J et al(1993) DNA Cell Biol 12:441–53).

VARIANTS/HOMOLOGUES/DERIVATIVES

In addition to the specific amino acid sequences and nucleotidesequences mentioned herein, the present invention also encompasses theuse of variants, homologue and derivatives thereof. Here, the term“homology” can be equated with “identity”.

In the present context, an homologous sequence is taken to include anamino acid sequence which may be at least 75, 85 or 90% identical,preferably at least 95 or 98% identical. Although homology can also beconsidered in terms of similarity (i.e. amino acid residues havingsimilar chemical properties/functions), in the context of the presentinvention it is preferred to express homology in terms of sequenceidentity.

Homology comparisons can be conducted by eye, or more usually, with theaid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate % homologybetween two or more sequences.

% homology may be calculated over contiguous sequences, i.e. onesequence is aligned with the other sequence and each amino acid in onesequence is directly compared with the corresponding amino acid in theother sequence, one residue at a time. This is called an “ungapped”alignment. Typically, such ungapped alignments are performed only over arelatively short number of residues.

Although this is a very simple and consistent method, it fails to takeinto consideration that, for example, in an otherwise identical pair ofsequences, one insertion or deletion will cause the following amino acidresidues to be put out of alignment, thus potentially resulting in alarge reduction in % homology when a global alignment is performed.Consequently, most sequence comparison methods are designed to produceoptimal alignments that take into consideration possible insertions anddeletions without penalising unduly the overall homology score. This isachieved by inserting “gaps” in the sequence alignment to try tomaximise local homology.

However, these more complex methods assign “gap penalties” to each gapthat occurs in the alignment so that, for the same number of identicalamino acids, a sequence alignment with as few gaps aspossible—reflecting higher relatedness between the two comparedsequences—will achieve a higher score than one with many gaps. “Affinegap costs” are typically used that charge a relatively high cost for theexistence of a gap and a smaller penalty for each subsequent residue inthe gap. This is the most commonly used gap scoring system. High gappenalties will of course produce optimised alignments with fewer gaps.Most alignment programs allow the gap penalties to be modified. However,it is preferred to use the default values when using such software forsequence comparisons. For example when using the GCG Wisconsin Bestfitpackage (see below) the default gap penalty for amino acid sequences is−12 for a gap and −4 for each extension.

Calculation of maximum % homology therefore firstly requires theproduction of an optimal alignment, taking into consideration gappenalties. A suitable computer program for carrying out such analignment is the GCG Wisconsin Bestfit package (University of Wisconsin,U.S.A.; Devereux et al., 1984, Nucleic Acids Research 12:387). Examplesof other software than can perform sequence comparisons include, but arenot limited to, the BLAST package (see Ausubel et al., 1999 ibid—Chapter18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403–410) and theGENEWORKS suite of comparison tools. Both BLAST and FASTA are availablefor offline and online searching (see Ausubel et al., 1999 ibid, pages7–58 to 7–60). However it is preferred to use the GCG Bestfit program.

A further useful reference is that found in FEMS Microbiol Lett May 15,1999;174(2):247–50 (and a published erratum appears in FEMS MicrobiolLett Aug. 1, 1999;177(1):187–8).

Although the final % homology can be measured in terms of identity, thealignment process itself is typically not based on an all-or-nothingpair comparison. Instead, a scaled similarity score matrix is generallyused that assigns scores to each pairwise comparison based on chemicalsimilarity or evolutionary distance. An example of such a matrixcommonly used is the BLOSUM62 matrix—the default matrix for the BLASTsuite of programs. GCG Wisconsin programs generally use either thepublic default values or a custom symbol comparison table if supplied(see user manual for further details). It is preferred to use the publicdefault values for the GCG package, or in the case of other software,the default matrix, such as BLOSUM62.

Once the software has produced an optimal alignment, it is possible tocalculate % homology, preferably % sequence identity. The softwaretypically does this as part of the sequence comparison and generates anumerical result.

The sequences may also have deletions, insertions or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent substance. Deliberate amino acid substitutionsmay be made on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the secondary binding activity of the substance isretained. For example, negatively charged amino acids include asparticacid and glutamic acid; positively charged amino acids include lysineand arginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values include leucine, isoleucine, valine,glycine, alanine, asparagine, glutamine, serine, threonine,phenylalanine, and tyrosine.

Conservative substitutions may be made, for example according to theTable below. Amino acids in the same block in the second column andpreferably in the same line in the third column may be substituted foreach other:

ALIPHATIC Non-polar G A P I L V Polar - uncharged C S T M N Q Polar -charged D E K R AROMATIC H F W Y

EXPRESSION VECTORS

The nucleotide sequence for use as the target or for expressing thetarget can be incorporated into a recombinant replicable vector. Thevector may be used to replicate and express the nucleotide sequence inand/or from a compatible host cell. Expression may be controlled usingcontrol sequences which include promoters/enhancers and other expressionregulation signals. Prokaryotic promoters and promoters functional ineukaryotic cells may be used. Tissue specific or stimuli specificpromoters may be used. Chimeric promoters may also be used comprisingsequence elements from two or more different promoters described above.

The protein produced by a host recombinant cell by expression of thenucleotide sequence may be secreted or may be contained intracellularlydepending on the sequence and/or the vector used. The coding sequencescan be designed with signal sequences which direct secretion of thesubstance coding sequences through a particular prokaryotic oreukaryotic cell membrane.

FUSION PROTEINS

The target amino acid sequence may be produced as a fusion protein, forexample to aid in extraction and purification. Examples of fusionprotein partners include glutathione-S-transferase (GST), 6xHis, GAL4(DNA binding and/or transcriptional activation domains) and(-galactosidase. It may also be convenient to include a proteolyticcleavage site between the fusion protein partner and the proteinsequence of interest to allow removal of fusion protein sequences.Preferably the fusion protein will not hinder the activity of thetarget.

The fusion protein may comprise an antigen or an antigenic determinantfused to the substance of the present invention. In this embodiment, thefusion protein may be a non-naturally occurring fusion proteincomprising a substance which may act as an adjuvant in the sense ofproviding a generalised stimulation of the immune system. The antigen orantigenic determinant may be attached to either the amino or carboxyterminus of the substance.

In another embodiment of the invention, the amino acid sequence may beligated to a heterologous sequence to encode a fusion protein. Forexample, for screening of peptide libraries for agents capable ofaffecting the substance activity, it may be useful to encode a chimericsubstance expressing a heterologous epitope that is recognised by acommercially available antibody.

THERAPY

The compounds of the present invention may be used as therapeuticagents—i.e. in therapy applications.

The term “therapy” includes curative effects, alleviation effects, andprophylactic effects.

The therapy may be on humans or animals, preferably female animals.

PHARMACEUTICAL COMPOSITIONS

In one aspect, the present invention provides a pharmaceuticalcomposition, which comprises a compound according to the presentinvention and optionally a pharmaceutically acceptable carrier, diluentor excipient (including combinations thereof).

The pharmaceutical compositions may be for human or animal usage inhuman and veterinary medicine and will typically comprise any one ormore of a pharmaceutically acceptable diluent, carrier, or excipient.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985).The choice of pharmaceutical carrier, excipient or diluent can beselected with regard to the intended route of administration andstandard pharmaceutical practice. The pharmaceutical compositions maycomprise as—or in addition to—the carrier, excipient or diluent anysuitable binder(s), lubricant(s), suspending agent(s), coating agent(s),solubilising agent(s).

Preservatives, stabilisers, dyes and even flavouring agents may beprovided in the pharmaceutical composition. Examples of preservativesinclude sodium benzoate, sorbic acid and esters of p-hydroxybenzoicacid. Antioxidants and suspending agents may be also used.

There may be different composition/formulation requirements dependent onthe different delivery systems. By way of example, the pharmaceuticalcomposition of the present invention may be formulated to be deliveredusing a mini-pump or by a mucosal route, for example, as a nasal sprayor aerosol for inhalation or ingestable solution, or parenterally inwhich the composition is formulated by an injectable form, for delivery,by, for example, an intravenous, intramuscular or subcutaneous route.Alternatively, the formulation may be designed to be delivered by bothroutes.

Where the agent is to be delivered mucosally through thegastrointestinal mucosa, it should be able to remain stable duringtransit though the gastrointestinal tract; for example, it should beresistant to proteolytic degradation, stable at acid pH and resistant tothe detergent effects of bile.

Where appropriate, the pharmaceutical compositions can be administeredby inhalation, in the form of a suppository or pessary, topically in theform of a lotion, solution, cream, ointment or dusting powder, by use ofa skin patch, orally in the form of tablets containing excipients suchas starch or lactose, or in capsules or ovules either alone or inadmixture with excipients, or in the form of elixirs, solutions orsuspensions containing flavouring or colouring agents, or they can beinjected parenterally, for example intravenously, intramuscularly orsubcutaneously. For parenteral administration, the compositions may bebest used in the form of a sterile aqueous solution which may containother substances, for example enough salts or monosaccharides to makethe solution isotonic with blood. For buccal or sublingualadministration the compositions may be administered in the form oftablets or lozenges which can be formulated in a conventional manner.

COMBINATION PHARMACEUTICAL

The compound of the present invention may be used in combination withone or more other active agents, such as one or more otherpharmaceutically active agents.

By way of example, the compounds of the present invention may be used incombination with other STS inhibitors and/or other inhibitors such as anaromatase inhibitor (such as for example, 4hydroxyandrostenedione(4-OHA)) and/or steroids—such as the naturally occurringsterneurosteroids dehydroepiandrosterone sulfate (DHEAS) andpregnenolone sulfate (PS) and/or other structurally similar organiccompounds. Examples of other STS inhibitors may be found in the abovereferences. By way of example, STS inhibitors for use in the presentinvention include EMATE, and either or both of the 2-ethyl and 2-methoxy17-deoxy compounds that are analogous to compound 5 presented herein.

In addition, or in the alternative, the compound of the presentinvention may be used in combination with a biological responsemodifier.

The term biological response modifier (“BRM”) includes cytokines, immunemodulators, growth factors, haematopoiesis regulating factors, colonystimulating factors, chemotactic, haemolytic and thrombolytic factors,cell surface receptors, ligands, leukocyte adhesion molecules,monoclonal antibodies, preventative and therapeutic vaccines, hormones,extracellular matrix components, fibronectin, etc. For someapplications, preferably, the biological response modifier is acytokine. Examples of cytokines include: interleukins (IL)—such as IL-1,IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,IL-19; Tumour Necrosis Factor (TNF)—such as TNF-α; Interferon alpha,beta and gamma; TGF-β. For some applications, preferably the cytokine istumour necrosis factor (TNF). For some applications, the TNF may be anytype of TNF—such as TNF-α, TNF-β, including derivatives or mixturesthereof. More preferably the cytokine is TNF-α. Teachings on TNF may befound in the art—such as WO-A-98/08870 and WO-A-98/13348.

ADMINISTRATION

Typically, a physician will determine the actual dosage which will bemost suitable for an individual subject and it will vary with the age,weight and response of the particular patient. The dosages below areexemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited.

The compositions of the present invention may be administered by directinjection. The composition may be formulated for parenteral, mucosal,intramuscular, intravenous, subcutaneous, intraocular or transdermaladministration. Depending upon the need, the agent may be administeredat a dose of from 0.01 to 30 mg/kg body weight, such as from 0.1 to 10mg/kg, more preferably from 0.1 to 1 mg/kg body weight.

By way of further example, the agents of the present invention may beadministered in accordance with a regimen of 1 to 4 times per day,preferably once or twice per day. The specific dose level and frequencyof dosage for any particular patient may be varied and will depend upona variety of factors including the activity of the specific compoundemployed, the metabolic stability and length of action of that compound,the age, body weight, general health, sex, diet, mode and time ofadministration, rate of excretion, drug combination, the severity of theparticular condition, and the host undergoing therapy.

Aside from the typical modes of delivery—indicated above—the term“administered” also includes delivery by techniques such as lipidmediated transfection, liposomes, immunoliposomes, lipofectin, cationicfacial amphiphiles (CFAs) and combinations thereof. The routes for suchdelivery mechanisms include but are not limited to mucosal, nasal, oral,parenteral, gastrointestinal, topical, or sublingual routes.

The term “administered” includes but is not limited to delivery by amucosal route, for example, as a nasal spray or aerosol for inhalationor as an ingestable solution; a parenteral route where delivery is by aninjectable form, such as, for example, an intravenous, intramuscular orsubcutaneous route.

Thus, for pharmaceutical administration, the STS inhibitors of thepresent invention can be formulated in any suitable manner utilisingconventional pharmaceutical formulating techniques and pharmaceuticalcarriers, adjuvants, excipients, diluents etc. and usually forparenteral administration. Approximate effective dose rates may be inthe range from 1 to 1000 mg/day, such as from 10 to 900 mg/day or evenfrom 100 to 800 mg/day depending on the individual activities of thecompounds in question and for a patient of average (70 Kg) bodyweight.More usual dosage rates for the preferred and more active compounds willbe in the range 200 to 800 mg/day, more preferably, 200 to 500 mg/day,most preferably from 200 to 250 mg/day. They may be given in single doseregimes, split dose regimes and/or in multiple dose regimes lasting overseveral days. For oral administration they may be formulated in tablets,capsules, solution or suspension containing from 100 to 500 mg ofcompound per unit dose. Alternatively and preferably the compounds willbe formulated for parenteral administration in a suitable parenterallyadministrable carrier and providing single daily dosage rates in therange 200 to 800 mg, preferably 200 to 500, more preferably 200 to 250mg. Such effective daily doses will, however, vary depending on inherentactivity of the active ingredient and on the bodyweight of the patient,such variations being within the skill and judgement of the physician.

CELL CYCLING

The compounds of the present invention may be useful in the method oftreatment of a cell cycling disorder.

As discussed in “Molecular Cell Biology” 3rd Ed. Lodish et al. pages177–181 different eukaryotic cells can grow and divide at quitedifferent rates. Yeast cells, for example, can divide every 120 min.,and the first divisions of fertilised eggs in the embryonic cells of seaurchins and insects take only 1530 min. because one large pre-existingcell is subdivided. However, most growing plant and animal cells take10–20 hours to double in number, and some duplicate at a much slowerrate. Many cells in adults, such as nerve cells and striated musclecells, do not divide at all; others, like the fibroblasts that assist inhealing wounds, grow on demand but are otherwise quiescent.

Still, every eukaryotic cell that divides must be ready to donate equalgenetic material to two daughter cells. DNA synthesis in eukaryotes doesnot occur throughout the cell division cycle but is restricted to a partof it before cell division.

The relationship between eukaryotic DNA synthesis and cell division hasbeen thoroughly analysed in cultures of mammalian cells that were allcapable of growth and division. In contrast to bacteria, it was found,eukaryotic cells spend only a part of their time in DNA synthesis, andit is completed hours before cell division (mitosis). Thus a gap of timeoccurs after DNA synthesis and before cell division; another gap wasfound to occur after division and before the next round of DNAsynthesis. This analysis led to the conclusion that the eukaryotic cellcycle consists of an M (mitotic) phase, a G₁ phase (the first gap), theS (DNA synthesis) phase, a G₂ phase (the second gap), and back to M. Thephases between mitoses (G₁, S, and G₂) are known collectively as theinterphase.

Many nondividing cells in tissues (for example, all quiescentfibroblasts) suspend the cycle after mitosis and just prior to DNAsynthesis; such “resting” cells are said to have exited from the cellcycle and to be in the G₀ state.

It is possible to identify cells when they are in one of the threeinterphase stages of the cell cycle, by using a fluorescence-activatedcell sorter (FACS) to measure their relative DNA content: a cell that isin G₁ (before DNA synthesis) has a defined amount x of DNA; during S(DNA replication), it has between x and 2x; and when in G₂ (or M), ithas 2x of DNA.

The stages of mitosis and cytokinesis in an animal cell are as follows

-   (a) Interphase. The G₂ stage of interphase immediately precedes the    beginning of mitosis. Chromosomal DNA has been replicated and bound    to protein during the S phase, but chromosomes are not yet seen as    distinct structures. The nucleolus is the only nuclear substructure    that is visible under light microscope. In a diploid cell before DNA    replication there are two morphologic chromosomes of each type, and    the cell is said to be 2n. In G₂, after DNA replication, the cell is    4n. There are four copies of each chromosomal DNA. Since the sister    chromosomes have not yet separated from each other, they are called    sister chromatids.-   b) Early prophase. Centrioles, each with a newly formed daughter    centriole, begin moving toward opposite poles of the cell; the    chromosomes can be seen as long threads. The nuclear membrane begins    to disaggregate into small vesicles.-   (c) Middle and late prophase. Chromosome condensation is completed;    each visible chromosome structure is composed of two chromatids held    together at their centromeres. Each chromatid contains one of the    two newly replicated daughter DNA molecules. The microtubular    spindle begins to radiate from the regions just adjacent to the    centrioles, which are moving closer to their poles. Some spindle    fibres reach from pole to pole; most go to chromatids and attach at    kinetochores.-   (d) Metaphase. The chromosomes move toward the equator of the cell,    where they become aligned in the equatorial plane. The sister    chromatids have not yet separated.-   (e) Anaphase. The two sister chromatids separate into independent    chromosomes. Each contains a centromere that is linked by a spindle    fibre to one pole, to which it moves. Thus one copy of each    chromosome is donated to each daughter cell. Simultaneously, the    cell elongates, as do the pole-to-pole spindles. Cytokinesis begins    as the cleavage furrow starts to form.-   (f) Telophase. New membranes form around the daughter nuclei; the    chromosomes uncoil and become less distinct, the nucleolus becomes    visible again, and the nuclear membrane forms around each daughter    nucleus. Cytokinesis is nearly complete, and the spindle disappears    as the microtubules and other fibres depolymerise. Throughout    mitosis the “daughter” centriole at each pole grows until it is    full-length. At telophase the duplication of each of the original    centrioles is completed, and new daughter centrioles will be    generated during the next interphase.-   (g) Interphase. Upon the completion of cytokinesis, the cell enters    the G₁ phase of the cell cycle and proceeds again around the cycle.

It will be appreciated that cell cycling is an extremely important cellprocess. Deviations from normal cell cycling can result in a number ofmedical disorders. Increased and/or unrestricted cell cycling may resultin cancer. Reduced cell cycling may result in degenerative conditions.Use of the compound of the present invention may provide a means totreat such disorders and conditions.

Thus, the compound of the present invention may be suitable for use inthe treatment of cell cycling disorders such as cancers, includinghormone dependent and hormone independent cancers.

In addition, the compound of the present invention may be suitable forthe treatment of cancers such as breast cancer, ovarian cancer,endometrial cancer, sarcomas, melanomas, prostate cancer, pancreaticcancer etc. and other solid tumours.

For some applications, cell cycling is inhibited and/or prevented and/orarrested, preferably wherein cell cycling is prevented and/or arrested.In one aspect cell cycling may be inhibited and/or prevented and/orarrested in the G₂/M phase. In one aspect cell cycling may beirreversibly prevented and/or inhibited and/or arrested, preferablywherein cell cycling is irreversibly prevented and/or arrested.

By the term “irreversibly prevented and/or inhibited and/or arrested” itis meant after application of a compound of the present invention, onremoval of the compound the effects of the compound, namely preventionand/or inhibition and/or arrest of cell cycling, are still observable.More particularly by the term “irreversibly prevented and/or inhibitedand/or arrested” it is meant that when assayed in accordance with thecell cycling assay protocol presented herein, cells treated with acompound of interest show less growth after Stage 2 of the protocol 1than control cells. Details on this protocol are presented below.

Thus, the present invention provides compounds which: cause inhibitionof growth of oestrogen receptor positive (ER+) and ER negative (ER−)breast cancer cells in vitro by preventing and/or inhibiting and/orarresting cell cycling; and/or cause regression of nitroso-methyl urea(NMU)-induced mammary tumours in intact animals (i.e. notovariectomised), and/or prevent and/or inhibit and/or arrest cellcycling in cancer cells; and/or act in vivo by preventing and/orinhibiting and/or arresting cell cycling and/or act as a cell cyclingagonist.

CELL CYCLING ASSAY (PROTOCOL 6) Procedure

Stage 1

MCF-7 breast cancer cells are seeded into multi-well culture plates at adensity of 105 cells/well. Cells were allowed to attach and grown untilabout 30% confluent when they are treated as follows:

-   Control—no treatment-   Compound of Interest (COI) 20 μM

Cells are grown for 6 days in growth medium containing the COI withchanges of medium/COI every 3 days. At the end of this period cellnumbers were counted using a Coulter cell counter.

Stage 2

After treatment of cells for a 6-day period with the COI cells arere-seeded at a density of 10⁴ cells/well. No further treatments areadded. Cells are allowed to continue to grow for a further 6 days in thepresence of growth medium. At the end of this period cell numbers areagain counted.

CANCER

As indicated, the compounds of the present invention may be useful inthe treatment of a cell cycling disorder. A particular cell cyclingdisorder is cancer.

Cancer remains a major cause of mortality in most Western countries.Cancer therapies developed so far have included blocking the action orsynthesis of hormones to inhibit the growth of hormone-dependenttumours. However, more aggressive chemotherapy is currently employed forthe treatment of hormone-independent tumours.

Hence, the development of a pharmaceutical for anti-cancer treatment ofhormone dependent and/or hormone independent tumours, yet lacking someor all of the side-effects associated with chemotherapy, would representa major therapeutic advance.

It is known that oestrogens undergo a number of hydroxylation andconjugation reactions after their synthesis. Until recently it wasthought that such reactions were part of a metabolic process thatultimately rendered oestrogens water soluble and enhanced theirelimination from the body. It is now evident that some hydroxymetabolites (e.g. 2-hydroxy and 16alpha-hydroxy) and conjugates (e.g.oestrone sulphate, E1S) are important in determining some of the complexactions that oestrogens have in the body.

Workers have investigated the formation of 2- and 16-hydroxylatedoestrogens in relation to conditions that alter the risk of breastcancer. There is now evidence that factors which increase 2-hydroxylaseactivity are associated with a reduced cancer risk, while thoseincreasing 16alpha-hydroxylation may enhance the risk of breast cancer.Further interest in the biological role of estrogen metabolites has beenstimulated by the growing body of evidence that 2-methoxyoestradiol isan endogenous metabolite with anti-mitotic properties. 2-MeOE2 is formedfrom 2-hydroxy estradiol (2-OHE2) by catechol estrogen methyltransferase, an enzyme that is widely distributed throughout the body.

Workers have shown that in vivo 2-MeOE2 inhibits the growth of tumoursarising from the subcutaneous injection of Meth A sarcoma, B16 melanomaor MDA-MB-435 estrogen receptor negative (ER−) breast cancer cells. Italso inhibits endothelial cell proliferation and migration, and in vitroangiogenesis. It was suggested that the ability of 2-MeOE2 to inhibittumour growth in vivo may be due to its ability to inhibittumour-induced angiogenesis rather than direct inhibition of theproliferation of tumour cells.

The mechanism by which 2-MeOE2 exerts its potent anti-mitogenic andanti-angiogenic effects is still being elucidated. There is evidencethat at high concentrations it can inhibit microtubule polymerisationand act as a weak inhibitor of colchicine binding to tubulin. Recently,however, at concentrations that block mitosis, tubulin filaments incells were not found to be depolymerised but to have an identicalmorphology to that seen after taxol treatment. It is possible,therefore, that like taxol, a drug that is used for breast and ovarianbreast cancer therapy, 2-MeOE2 acts by stabilising microtubule dynamics.

While the identification of 2-MeOE2 as a new therapy for cancerrepresents an important advance, the bioavailability of orallyadministered oestrogens is poor. Furthermore, they can undergo extensivemetabolism during their first pass through the liver. As part of aresearch programme to develop a steroid sulphatase inhibitor for breastcancer therapy, oestrone-3-O-sulphamate (EMATE) was identified as apotent active site-directed inhibitor. Unexpectedly, EMATE proved topossess potent oestrogenic properties with its oral uterotrophicactivity in rats being a 100-times higher than that of estradiol. Itsenhanced oestrogenicity is thought to result from its absorption by redblood cells (rbcs) which protects it from inactivation during itspassage through the liver and which act as a reservoir for its slowrelease for a prolonged period of time. A number of A-ring modifiedanalogues were synthesised and tested, including2-methoxyoestrone-3-O-sulphamate. While this compound was equipotentwith EMATE as a steroid sulphatase inhibitor, it was devoid ofoestrogenicity.

We believe that the compound of the present invention provides a meansfor the treatment of cancers and, especially, breast cancer.

In addition or in the alternative the compound of the present inventionmay be useful in the blocking the growth of cancers including leukaemiasand solid tumours such as breast, endometrium, prostate, ovary andpancreatic tumours.

THERAPY CONCERNING OESTROGEN

We believe that some of the compounds of the present invention may beuseful in the control of oestrogen levels in the body—in particular infemales. Thus, some of the compounds may be useful as providing a meansof fertility control—such as an oral contraceptive tablet, pill,solution or lozenge. Alternatively, the compound could be in the form ofan implant or as a patch.

Thus, the compounds of the present invention may be useful in treatinghormonal conditions associated with oestrogen.

In addition or in the alternative the compound of the present inventionmay be useful in treating hormonal conditions in addition to thoseassociated with oestrogen. Hence, the compound of the present inventionmay also be capable of affecting hormonal activity and may also becapable of affecting an immune response.

NEURODEGENERATIVE DISEASES

We believe that some of the compounds of the present invention may beuseful in the treatment of neurodenerative diseases, and similarconditions.

By way of example, it is believed that STS inhibitors may be useful inthe enhancing the memory function of patients suffering from illnessessuch as amnesia, head injuries, Alzheimer's disease, epileptic dementia,presenile dementia, post traumatic dementia, senile dementia, vasculardementia and post-stroke dementia or individuals otherwise seekingmemory enhancement.

TH1

We believe that some of the compounds of the present invention may beuseful in TH1 implications.

By way of example, it is believed that the presence of STS inhibitorswithin the macrophage or other antigen presenting cells may lead to adecreased ability of sensitised T cells to mount a TH1 (high IL-2, IFNγlow IL-4) response. The normal regulatory influence of other steroidssuch as glucocorticoids would therefore predominate.

INFLAMATORY CONDITIONS

We believe that some of the compounds of the present invention may beuseful in treating inflammatory conditions—such as conditions associatedwith any one or more of: autoimmunity, including for example, rheumatoidarthritis, type I and II diabetes, systemic lupus erythematosus,multiple sclerosis, myasthenia gravis, thyroiditis, vasculitis,ulcerative colitis and Crohn's disease, skin disorders e.g. psoriasisand contact dermatitis; graft versus host disease; eczema; asthma andorgan rejection following transplantation.

By way of example, it is believed that STS inhibitors may prevent thenormal physiological effect of DHEA or related steroids on immune and/orinflammatory responses.

The compounds of the present invention may be useful in the manufactureof a medicament for revealing an endogenous glucocorticoid-like effect.

OTHER THERAPIES

It is also to be understood that the compound/composition of the presentinvention may have other important medical implications.

For example, the compound or composition of the present invention may beuseful in the treatment of the disorders listed in WO-A-99/52890—viz:

In addition, or in the alternative, the compound or composition of thepresent invention may be useful in the treatment of the disorders listedin WO-A-98/05635. For ease of reference, part of that list is nowprovided: cancer, inflammation or inflammatory disease, dermatologicaldisorders, fever, cardiovascular effects, haemorrhage, coagulation andacute phase response, cachexia, anorexia, acute infection, HIVinfection, shock states, graft-versus-host reactions, autoimmunedisease, reperfusion injury, meningitis, migraine and aspirin-dependentanti-thrombosis; tumour growth, invasion and spread, angiogenesis,metastases, malignant, ascites and malignant pleural effusion; cerebralischaemia, ischaemic heart disease, osteoarthritis, rheumatoidarthritis, osteoporosis, asthma, multiple sclerosis, neurodegeneration,Alzheimer's disease, atherosclerosis, stroke, vasculitis, Crohn'sdisease and ulcerative colitis; periodontitis, gingivitis; psoriasis,atopic dermatitis, chronic ulcers, epidermolysis bullosa; cornealulceration, retinopathy and surgical wound healing; rhinitis, allergicconjunctivitis, eczema, anaphylaxis; restenosis, congestive heartfailure, endometriosis, atherosclerosis or endosclerosis.

In addition, or in the alternative, the compound or composition of thepresent invention may be useful in the treatment of disorders listed inWO-A-98/07859. For ease of reference, part of that list is now provided:cytokine and cell proliferation/differentiation activity;immunosuppressant or immunostimulant activity (e.g. for treating immunedeficiency, including infection with human immune deficiency virus;regulation of lymphocyte growth; treating cancer and many autoimmunediseases, and to prevent transplant rejection or induce tumourimmunity); regulation of haematopoiesis, e.g. treatment of myeloid orlymphoid diseases; promoting growth of bone, cartilage, tendon, ligamentand nerve tissue, e.g. for healing wounds, treatment of burns, ulcersand periodontal disease and neurodegeneration; inhibition or activationof follicle-stimulating hormone (modulation of fertility);chemotactic/chemokinetic activity (e.g. for mobilising specific celltypes to sites of injury or infection); haemostatic and thrombolyticactivity (e.g. for treating haemophilia and stroke); antiinflammatoryactivity (for treating e.g. septic shock or Crohn's disease); asantimicrobials; modulators of e.g. metabolism or behaviour; asanalgesics; treating specific deficiency disorders; in treatment of e.g.psoriasis, in human or veterinary medicine.

In addition, or in the alternative, the composition of the presentinvention may be useful in the treatment of disorders listed inWO-A-98/09985. For ease of reference, part of that list is now provided:macrophage inhibitory and/or T cell inhibitory activity and thus,anti-inflammatory activity; anti-immune activity, i.e. inhibitoryeffects against a cellular and/or humoral immune response, including aresponse not associated with inflammation; inhibit the ability ofmacrophages and T cells to adhere to extracellular matrix components andfibronectin, as well as up-regulated fas receptor expression in T cells;inhibit unwanted immune reaction and inflammation including arthritis,including rheumatoid arthritis, inflammation associated withhypersensitivity, allergic reactions, asthma, systemic lupuserythematosus, collagen diseases and other autoimmune diseases,inflammation associated with atherosclerosis, arteriosclerosis,atherosclerotic heart disease, reperfusion injury, cardiac arrest,myocardial infarction, vascular inflammatory disorders, respiratorydistress syndrome or other cardiopulmonary diseases, inflammationassociated with peptic ulcer, ulcerative colitis and other diseases ofthe gastrointestinal tract, hepatic fibrosis, liver cirrhosis or otherhepatic diseases, thyroiditis or other glandular diseases,glomerulonephritis or other renal and urologic diseases, otitis or otheroto-rhino-laryngological diseases, dermatitis or other dermal diseases,periodontal diseases or other dental diseases, orchitis orepididimo-orchitis, infertility, orchidal trauma or other immune-relatedtesticular diseases, placental dysfunction, placental insufficiency,habitual abortion, eclampsia, pre-eclampsia and other immune and/orinflammatory-related gynaecological diseases, posterior uveitis,intermediate uveitis, anterior uveitis, conjunctivitis, chorioretinitis,uveoretinitis, optic neuritis, intraocular inflammation, e.g. retinitisor cystoid macular oedema, sympathetic ophthalmia, scleritis, retinitispigmentosa, immune and inflammatory components of degenerative fondusdisease, inflammatory components of ocular trauma, ocular inflammationcaused by infection, proliferative vitreo-retinopathies, acute ischaemicoptic neuropathy, excessive scarring, e.g. following glaucoma filtrationoperation, immune and/or inflammation reaction against ocular implantsand other immune and inflammatory-related ophthalmic diseases,inflammation associated with autoimmune diseases or conditions ordisorders where, both in the central nervous system (CNS) or in anyother organ, immune and/or inflammation suppression would be beneficial,Parkinson's disease, complication and/or side effects from treatment ofParkinson's disease, AIDS-related dementia complex HIV-relatedencephalopathy, Devic's disease, Sydenham chorea, Alzheimer's diseaseand other degenerative diseases, conditions or disorders of the CNS,inflammatory components of stokes, post-polio syndrome, immune andinflammatory components of psychiatric disorders, myelitis,encephalitis, subacute sclerosing pan-encephalitis, encephalomyelitis,acute neuropathy, subacute neuropathy, chronic neuropathy,Guillaim-Barre syndrome, Sydenham chora, myasthenia gravis,pseudo-tumour cerebri, Down's Syndrome, Huntington's disease,amyotrophic lateral sclerosis, inflammatory components of CNScompression or CNS trauma or infections of the CNS, inflammatorycomponents of muscular atrophies and dystrophies, and immune andinflammatory related diseases, conditions or disorders of the centraland peripheral nervous systems, post-traumatic inflammation, septicshock, infectious diseases, inflammatory complications or side effectsof surgery, bone marrow transplantation or other transplantationcomplications and/or side effects, inflammatory and/or immunecomplications and side effects of gene therapy, e.g. due to infectionwith a viral carrier, or inflammation associated with AIDS, to suppressor inhibit a humoral and/or cellular immune response, to treat orameliorate monocyte or leukocyte proliferative diseases, e.g. leukaemia,by reducing the amount of monocytes or lymphocytes, for the preventionand/or treatment of graft rejection in cases of transplantation ofnatural or artificial cells, tissue and organs such as cornea, bonemarrow, organs, lenses, pacemakers, natural or artificial skin tissue.

COMPOUND PREPARATION

The compounds of the present invention may be prepared by reacting anappropriate alcohol with a suitable chloride. By way of example, thesulphamate compounds of the present invention may be prepared byreacting an appropriate alcohol with a suitable sulfamoyl chloride, ofthe formula R⁴R⁵NSO₂Cl.

Typical conditions for carrying out the reaction are as follows.

Sodium hydride and a sulfamoyl chloride are added to a stirred solutionof the alcohol in anhydrous dimethyl formamide at 0° C. Subsequently,the reaction is allowed to warm to room temperature whereupon stirringis continued for a further 24 hours. The reaction mixture is poured ontoa cold saturated solution of sodium bicarbonate and the resultingaqueous phase is extracted with dichloromethane. The combined organicextracts are dried over anhydrous MgSO₄. Filtration followed by solventevaporation in vacuo and co-evaporated with toluene affords a cruderesidue which is further purified by flash chromatography.

Preferably, the alcohol is derivatised, as appropriate, prior toreaction with the sulfamoyl chloride. Where necessary, functional groupsin the alcohol may be protected in known manner and the protecting groupor groups removed at the end of the reaction.

Preferably, the sulphamate compounds are prepared according to theteachings of Page et al (1990 Tetrahedron 46; 2059–2068).

The phosphonate compounds may be prepared by suitably combining theteachings of Page et al (1990 Tetrahedron 46; 2059–2068) andPCT/GB92/01586.

The sulphonate compounds may be prepared by suitably adapting theteachings of Page et al (1990 Tetrahedron 46; 2059–2068) andPCT/GB92/01586.

The thiophosphonate compounds may be prepared by suitably adapting theteachings of Page et al (1990 Tetrahedron 46; 2059–2068) andPCT/GB91/00270.

Preferred preparations are also presented in the following text.

SUMMARY

In summation, the present invention provides novel compounds for use assteroid sulphatase inhibitors and/or modulators of apoptosis and/ormodulators of cell cycling and/or cell growth, and pharmaceuticalcompositions containing them.

EXAMPLES

The present invention will now be described only by way of example.However, it is to be understood that the examples also present preferredcompounds of the present invention, as well as preferred routes formaking same and useful intermediates in the preparation of same.

2-Methoxyestradiol derivatives and3,17β-dihydroxy-17α-(2-picolyl)-estra-1,3,5(10)-triene BLE01022BACKGROUND

Potent inhibition of steroid sulfatase activity by 3-O-sulfamate17α-benzyl (or 4′-tert-butylbenzyl)estra-1,3,5(10)-trienes has beenobserved by D. Poirier et al. (J. Med. Chem., 42, 2280–2286, 1999).Compound 2 inhibited the steroid sulfatase activity in intacttransfected (293) cell culture assays by inactivating the enzymeactivity. Compound 2 also inactivates the steroid sulfatase activity atlower concentration than EMATE in microsomes of transfected (293) cells.In this assay, an excess of natural substrate E₁S protects enzymeagainst inactivation by 2 or EMATE. Furthermore, the unsulfamoylatedanalogue of 2, compound 1, did not inactivate the steroid sulfatase.

Starting from 2-methoxyoestrone GSS33, the tertiary alcohols BLE01008,BLE01010 and BLE01014 were obtained by a stereoselective addition ofGrignard reagents to the C17-carbonyl (see also D. Poirier and R. P.Boivin, Bioorg. Med. Chem. Lett., 8, 1891–1896, 1998). The 18-methyl onthe β-face of steroids directs the nucleophilic attack on the lesshindered α-face, giving the 17α-alkylated compound. An excess ofGrignard and no protective groups were used. To facilitate thechromatographic separation of the tertiary alcohols from the startingmaterial 2-methoxyoestrone (of polarity very close of the tertiaryalcohols), a quantitative carbonyl reduction was carried out with NaBH₄(MeOH, 30 min, 0° C.) affording 2-methoxyestradiol. The17-α-(2-picolyl)-17-β-estradiol BLE01022 was obtained in 30% yield byreacting a solution of oestrone in THF with a solution of2-picollyllithium 1.8 M in cyclohexane/Et₂O. The HCl salt BLE01024 of17-α-(2-picolyl)-17-β-estradiol BLE01022 is in preparation.

The sulfamoylation proceed regioselectively at position C3, by adding asolution of sulfamoyl chloride about 0.7 M in toluene to a solution of3-hydroxysteroids BLE01008 and BLE01014 in DMF or dichloromethane andusing DBMP as base, yielding to the sulfamates BLE01016B and BLE01018.The low yields of the sulfamates BLE01016B and BLE01018 may be due tothe influence of the 17-α-hydroxy group and the steric hindrance of themethoxy group. Sulfamoylation of BLE01010 is in progress.

EXPERIMENTAL PART3,17β-Dihydroxy-17α-(4-tert-butylbenzyl)-2-methoxyestra-1,3,5(10)-trieneBLE01008

a) Preparation of 4-tert-butylbenzylmagnesium bromide

Magnesium (1.49 g, 61.2 mmol) was added in a dry three necked flaskunder nitrogen atmosphere. 2 ml of dry Et₂O were added directly on themagnesium and a solution of 4-tert-butylbenzyl bromide (3.67 ml, 20mmol) in dry Et₂O (18 ml) was added dropwise (in about 30 min),temperature reached 33–35° C. (Et₂O reflux). The reaction was allowed tostir 45 min. b) The in situ generated Grignard reagent (10 ml, 10 mmol)was then added slowly via syringe to a solution of 2-methoxyoestroneGSS33 (300 mg, 1 mmol) in dry THF (27 ml) at room temperature undernitrogen atmosphere (temperature reached about 28° C.) and the reactionwas allowed to stir overnight. A saturated solution of NH₄Cl (50 ml) wasadded and the solution was extracted with AcOEt (3×50 ml), dried overMgSO₄, filtered and evaporated under reduced pressure. The obtainedcrude mixture was dissolved im MeOH (27 ml) and treated at O° C. withNaBH₄ in excess (75 mg) for 30 min. The reaction was quenched with water(50 ml) and MeOH was evaporated under vacuum, and the mixture extractedwith AcOEt (3×50 ml), dried over MgSO₄, filtered and evaporated underreduced pressure. Purification by flash chromatography (column Ø=2.5 cm,h=20 cm) using as eluent 150 ml hexane then AcOEt:hexane=2:8 afforded3,17β-dihydroxy-17α-(4-tert-butylbenzyl)-2-methoxyestra-1,3,5(10)-trieneBLE01008 as a white solid, (342 mg, 65% yield).

C₃₀H₄₀O₃

MW 448.64

Mp 223–225° C.

¹H NMR 400 MHz (CDCl₃): 0.98 (s, 3H, C-18-CH ³ ), 1.22–1.82 (m, 10Hincluding 1H exch. D₂O, 17β-OH), 1.33 (s, 9H, t-Bu-), 1.87–1.95 (m, 1H),1.96–2.06 (m, 1H), 2.18–2.28 (m, 1H), 2.29–2.38 (m, 1H), 2.65 and 2.89(2d of AB system, 2H, J=13.3 Hz, —CHPh), 2.72–2.82 (m, 2H, C-6-H), 3.87(s, 3H, MeO—), 5.44 (s, 1H exch. D₂O, —OH phenol), 6.66 (s, 1H, ArH),6.82 (s, 1H, ArH), 7.22 and 7.35 (2d of AB system, 4H, J=8.2 Hz, —CH ²Ph-t-Bu).

M/S m/z (+ve FAB, rel. int.): 448.2 (100, M⁺), 431.2 (51), 300.1 (78),147.0 (76), 133.0 (15).

M/S m/z (−ve FAB, rel. int.): 447.2 [100, (M-H)⁻], 329.0 (22), 276.0(25), 258.0 (25), 195.0 (24), 139.0 (26), 92.0 (17).

HRMS (+ve FAB) m/z calcd for C₃₀H₄₀O₃ M⁺ 448.297746, found 448.296936.

Rf 0.46 (hexane:AcOEt=3:2), SM Rf 0.40

3,17β-Dihydroxy-17α-benzyl-2-methoxyestra-1,3,5(10)-triene BLE01014

Same as BLE01008 except we used a solution of commercially available(Aldrich) phenylmagnesium bromide 2.0 M in THF (5 ml, 10 mmol). Samework-up. Purification by flash chromatography (column Ø=2.5 cm, h=20 cm)using as eluent 150 ml hexane then AcOEt:hexane=1:9 then 2:8 afforded3,17β-dihydroxy-17α-benzyl-2-methoxyestra-1,3,5(10)-triene BLE01014 as awhite solid, (194 mg, 49% yield) after drying to the vacuum pump.

C₂₆H₃₂O₃

MW 392.53

Mp 209–213° C.

¹H NMR 400 MHz (CDCl₃): 0.98 (s, 3H, C-18-CH ³ ), 1.24–1.82 (m, 10Hincluding 1H exch. D₂O, 17β-OH), 1.86–2.02 (m, 2H), 2.18–2.39 (m, 2H),2.68 and 2.93 (2d of AB system, 2H, J=13.3 Hz, —CH ² Ph), 2.72–2.88 (m,2H, C-6-H), 3.87 (s, 3H, MeO), 5.45 (s, 1H exch. D₂O, —OH phenol), 6.66(s, 1H, ArH), 6.82 (s, 1H, ArH), 7.22–7.38 (m, 5H, ArH).

M/S m/z (+ve FAB, rel. int.): 392.1 (100, M⁺), 375.1 (28), 301.1 (13),189.0 (11), 165.0 (12), 115.0 (11).

M/S m/z (−ve FAB, rel. int.): 391.1 [100, (M-H)⁻], 323.0 (34), 275.0(42), 258.0 (42), 195.0 (35), 139.0 (38), 92.0 (33).

HRMS (+ve FAB) m/z calcd for C₂₆H₃₂O₃ M⁺ 392.235145, found 392.234909.

Rf 0.38 (hexane:AcOEt=3:2), SM Rf 0.40

3,17β-Dihydroxy-17α-(3-bromobenzyl)-2-methoxyestra-1,3,5(10)-trieneBLE01010

a) Preparation of 4-tert-butylbenzylmagnesium bromide

Magnesium (1.49 g, 61.2 mmol) was added in a dry three necked flaskunder nitrogen atmosphere. 2 ml of dry Et₂O were added directly on themagnesium and a solution of 3-bromobenzylbromide (5.00 g, 20 mmol) indry Et₂O (18 ml) was added dropwise (in about 30 min), temperaturereached 33–35° C. (Et₂O reflux). The reaction was allowed to stir 45min.

b) Same as BLE01008 using a solution of 3-bromobenzylmagnesium bromide(10.0 ml, 10 mmol) in Et₂O. Same work-up. Purification by flashchromatography (column Ø=2.5 cm, h=20 cm) using as eluent 150 ml hexanethen AcOEt:hexane=1:9 (500 ml) then 2:8 (500 ml) afforded3,17β-dihydroxy-17α-(3-bromobenzyl)-2-methoxyestra-1,3,5(10)-triene BLE01010 as a white solid, (98 mg, 18% yield) after drying to the vacuumpump.

C₂₆H₃₁BrO₃

MW 471.43

Mp 173–177° C.

¹H NMR 400 MHz (CDCl₃): 0.97 (s, 3H, C-18-CH ³ ), 1.22–1.82 (m, 10Hincluding 1H exch. D₂O, 17β-OH), 1.87–2.02 (m, 2H), 2.17–2.38 (m, 2H),2.62 and 2.91 (2d of AB system, 2H, J=13.7 Hz, —CH ² PhBr), 2.72–2.88(m, 2H, C-6-H), 3.87 (s, 3H, MeO—), 5.44 (s, 1H exch. D₂O, —OH phenol),6.66 (s, 1H, ArH), 6.81 (s, 1H, ArH), 7.16–7.42 (m, 3H, ArH), 7.47 (s,1H, ArH).

HRMS (+ve FAB) m/z calcd for C₂₆H₃₁BrO₃ M⁺ 470.145656, found 470.144646.

Rf 0.38 (hexane:AcOEt=3:2), SM Rf 0.40

3,17β-Dihydroxy-17α-(2-picolyl)-estra-1,3,5(10)-triene BLE01022

a) Preparation of 2-picolyllithium

In a 100 ml three neck round-bottom flask equipped with a refluxcondenser, thermometer and under nitrogen atmosphere a solution ofphenyllithium 1.8 M in cyclohexane/Et₂O=70/30 was introduced via syringe(20.5 ml, 36.98 mmol). Freshly distilled 2-picoline (3.65 ml, 36.98mmol) was added drop wise over a 5 min period (temperature reached to45° C.) and the dark red solution was kept over 40° C. (water bath) for30 min.

b) The mixture was cooled at −6° C. (ice-salt-water bath) and a solutionof oestrone (1.0 g, 3.70 mmol) in 27 ml of anhydrous THF was added. Thedark red solution was stirred at room temperature for 1h30. The mixturewas poured on ice-water (300 ml) and extracted with AcOEt (3×150 ml),dried over magnesium sulfate, filtered and evaporated under reducepressure. Purification by flash chromatography (column Ø=2.5 cm, h=20cm) using as eluent AcOEt:hexane=30:70 to 40:60 (900 ml) afforded3,17β-dihydroxy-17α-(2-picolyl)-estra-1,3,5(10)-triene BLE 01022 as awhite solid, (405 mg, 30% yield) after drying to the vacuum pump.

C₂₄H₂₉NO₂

MW 363.49

Mp 250–255° C.

¹H NMR 400 MHz (CD₃OD+DMSO-D₆): 0.95 (s, 3H, C-18-CH ³ ), 1.27–1.82 (m,9H), 1.86-1.95 (m, 2H), 2.12–2.21 (m, 1H), 2,28–2.36 (m, 1H), 2.70–2.85(m, 2H, C-6-H), 2.92 and 3.07 (2d of AB system, 2H, J=13.7 Hz, —CH ²C₅H₅N), 6.48 (s, 1H, C-4), 6.53 (d, 1H, J=8.6 Hz, C-2), 7.08 (d, 1H,J=8.6 Hz, C-1), 7.27 (t, 1H, J=6.2 Hz, C-4 pyr), 7.44 (d, 1H, J=7.4 Hz,C-3 pyr), 7.75 (t, 1H, J=7.8 Hz, C-5 pyr), 8.45 (d, 1H, J=5.1 Hz, C-6pyr), 2 OH not seen.

HRMS (+ve FAB) m/z calcd for C₂₄H₂₉NO₂ in progress.

3-O-Sulfamate17β-hydroxy-17α-(4-tert-butylbenzyl)-2-methoxyestra-1,3,5(10) -trieneBLE01016

To a stirred solution of 17α-α-(4-tert-butylbenzyl)-2-methoxyestradiolBLE01008 (182 mg, 0.41 mmol) in CH₂Cl₂ anhydrous (30 ml) was added2,6-di-tert-butyl-4-methylpyridine (DBMP) (250 mg, 1.22 mmol), and themixture was stirred under nitrogen atmosphere for 15 min at roomtemperature. A solution of sulfamoyl chloride about 0.7 M in toluene wasadded via syringe (3.48 ml, 2.43 mmol). The mixture was stirred 16 h atroom temperature. 50 ml of AcOEt was added and the solution was washedwith brine (3×50 ml), dried over MgSO₄, filtered and the solvent wasevaporated under vacuum. The crude mixture was purified by flashchromatography (column Ø=2.5 cm, h=20 cm) using as eluent 150 ml hexanethen acetone:hexane=25:75 to give unreacted phenol BLE01008 and3-O-sulfamate17β-hydroxy-17α-(4-tert-butylbenzyl)-2-methoxyestra-1,3,5(10)-trieneBLE01016 as a white solid, (70 mg, 33% yield) after drying to the vacuumpump.

C₃₀H₄₁NO₅S

MW 527.72

Mp 106–110° C.

¹H NMR 400 MHz (CDCl₃): 0.98 (s, 3H, C-18-CH ³ ), 1.22–1.82 (m, 10Hincluding 1H exch. D₂O, 17β-OH), 1.33 (s, 9H, t-Bu), 1.90–2.06 (m, 2H),2.22–2.39 (m, 2H), 2.64 and 2.89 (2d of AB system, 2H, J=12.9 Hz, —CH ²Ph-t-Bu), 2.78–2.84 (m, 2H, C-6-H), 3.89 (s, 3H, MeO), 4.95 (s, 2H exch.D₂O, —NH ² ), 6.96 (s, 1H, ArH), 7.06 (s, 1H, ArH), 7.21 and 7.35 (2d ofAB system, J=8.2 Hz, 4H, C₆H₄-t-Bu).

M/S m/z (+ve FAB, rel. int.): in progress

HRMS (+ve FAB) m/z calcd for C₃₀H₄₁NO₅S M⁺, found in progress.

Rf 0.12 (hexane:AcOEt=3:2), SM Rf 0.46

3-O-Sulfamate 17β-hydroxy-17α-benzyl-2-methoxyestra-1,3,5(10)-trieneBLE01018

To a stirred solution of 17α-benzyl-2-methoxyestradiol BLE01014 (171 mg,0.43 mmol) in DMF anhydrous (12 ml) was added DBMP (268 mg, 1.31 mmol),and the mixture was stirred under nitrogen atmosphere for 15 min at roomtemperature. A solution of sulfamoyl chloride about 0.7 M in toluene wasadded via syringe (3.84 ml, 2.61 mmol). The mixture was stirred 5 h atroom temperature. 50 ml of AcOEt was added and the solution was washedwith brine (3×50 ml), dried over MgSO₄, filtered and the solvent wasevaporated under vacuum. The crude mixture was purified by flashchromatography (column Ø=2.5 cm, h=20 cm) using as eluentacetone:hexane=20:80 to give unreacted phenol BLE01014 (73 mg, 43%) and3-O-sulfamate 17β-hydroxy-17α-benzyl-2-methoxyestra-1,3,5(10)-trieneBLE01018 as a white solid, (30 mg, 15% yield) after drying to the vacuumpump.

C₂₆H₃₃NO₅S

MW 471.61

Mp 174–177° C.

¹H NMR 400 MHz (CDCl₃): 0.98 (s, 3H, C-18-CH ³ ), 1.22–1.85 (m, 10Hincluding 1H exch. D₂O, 17β-OH), 1.90–2.04 (m, 2H), 2.22–2.38 (m, 2H),2.67 and 2.93 (2d of AB system, 2H, J=12.9 Hz, —CH ² Ph), 2.78–2.87 (m,2H, C-6-H), 3.89 (s, 3H, MeO), 4.97 (s, 2H exch. D₂O, —NH ² ), 6.96 (s,1H, ArH), 7.06 (s, 1H, ArH), 7.24–7.38 (m, J=8.2 Hz, C₆H₅—).

HPLC Waters: eluent MeOH:H₂O=80:20, flow 3 ml/min, Column WatersRadialpak C18 8×100 mm, detection 280 nm, BLE01018 RT=4.10 min 97.85%,impurity RT=9.69 min, 3.15%.

M/S m/z (+ve FAB, rel. int.): in progress

HRMS (+ve FAB) m/z calcd for C₂₆H₃₃NO₅S M⁺, found in progress.

Rf 0.08 (hexane:AcOEt=3:2), SM Rf 0.38

Biological Data

A number of compounds indentified in the tables below were tested.

Compound X Y Z 1 OSO₂NH₂ SMe β-OH, α-Bn 2 OH SMe β-OH, α-4-(tBu)Bn 3OSO₂NH₂ SMe β-OH, α-4-(tBu)Bn 4 OH SMe CH₂ 5 OSO₂NH₂ SMe CH₂ 6 OH SMeC═O 7 OSO₂NH₂ SMe C═O 8 OH SEt C═O 9 OSO₂NH₂ SEt C═O

The STS inhibition provided by the compounds was determined inaccordance with a plate assay and the Protocols set out herein.

Placental Microsomes, % inhibition Plate Assay, % inhibition Compound 10μM μM 100 nM 10 nM IC50 nM 0.1 μM 1 μM 10 μM 1 97.4 93.0 69.7 15.5  44<1 <1 31 2 — — — — — <1 <1 50 3 86.4 81.8 56.0 17.6  80 <1 <1 48 6 — — —— — <1 1 11  7* — 86.9 — 5.4 120 3 53 55 8 — — — — <1 <4 14 9 — 66.0 —<5 — <1 1 16 EMATE 99.5 97.8 85.1 27.3  18 — — — *IC50 2.5 nM T47Dcells.

T47D cell Placental growth, % Microsomes Plate assay inhibition %inhibition % inhibition R X Y Cpd Code STX 10 μM 10 nM 1 μM 10 nM 0.1 μM1 μM 10 μM OH H C-β-OSO₂NH₂, α-H BLE99031A  48 5.1¹ 0.5¹  21  24 46 OH HC-β-OSO₂NHAc, α-H BLE99058  42 7.8¹ 0.9¹  4  1 20 OH H C-β-OSO₂N(Me)₂,α-H BLE99067  43 8.7¹ −0.1¹  −5  −1 50 OH H C-β-OSO₂N(pentyl)₂, α-HBLE99073  22 −11.8² 1.2² OH H C-α-OSO₂N(pentyl)₂, β-H BLE00082  23 −22.0−28.0  6  24  7 OH H C-β-OSO₂N(Bn)₂, α-H BLE99060  40 1.36² −3.2² OH HC-α-OSO₂N(Bn)₂, β-H BLE99066  41 4.5¹ 1.4¹  11  5 27 OSO₂NH₂ H C-β-OH,α-H BLE00084  28  4  14 23 OSO₂NH₂ H C-β-OSO₂NH₂, α-H BLE99031B  49 IC5020 nM  7  12 32 OSO₂NH₂ H C-β-OSO₂NHAc, α-H BLE99065  46 91.0¹ 9.8¹  7 5 23 OSO₂NH₂ H C-β-OSO₂N(Me)₂, α-H BLE99069  47 96.3¹ 30.3¹  3  10 73OSO₂NH₂ H C-β-OSO₂N(pentyl)₂, α-H BLE99074 23.7² −9.3² OSO₂NH₂ HC-α-OSO₂N(pentyl)₂, β-H BLE00083B  25 52.5 −9.3  8  1 24 OSO₂NH₂ HC-β-OSO₂N(Bn)₂, α-H BLE99063  44 38.2¹ −1.9¹  0  15 69 OSO₂NH₂ HC-α-OSO₂N(Bn)₂, β-H BLE99068  45 58.2¹ 4.9¹ −18  −1 19 OH OMe C-β-OH,α-Bn BLE01014  98 −14 −14 ND OSO₂NH₂ OMe C-β-OH, α-Bn BLE01018 100 IC50430 nM −19 −12 ND OH OMe C-β-OH, α-(4′-^(t)Bu)Bn BLE01008  99 −12 −15 NDOSO₂NH₂ OMe C-β-OH, α-(4′-^(t)Bu)Bn BLE01016 101 IC50 4300 nM −14 −10 −3OSO₂NH₂ OMe C-β-OSO₂NH₂, α-H IC50 32 nM  7  12 32 OSO₂NH₂ OEtC-β-OSO₂NH₂, α-H IC50 1 μM  7  12 32 BLE00086  26 93.2  8  3 14 2-MeOE1 6  7 73 2-MOEMATE  15  42 88 2-MeOE2  −2  19 66 2-EtE1  −1  −2 662-EtEMATE  36  51 60 Taxol  46  96 91 ¹667COUMATE 1 μM 99.3, 10 nM 89.9;EMATE 1 μM 98.0, 10 nM 56.1 ²EMATE 1 μM 96.9, 10 nM 30.4-ve % meansstimulation

The invention will now be further described by the following numberedparagraphs:

-   1. A compound comprising a steroidal ring system and a group R¹    selected from any one of a sulphamate group, a phosphonate group, a    thiophosphonate group, a sulphonate group or a sulphonamide group;    wherein the D ring of the steroidal ring system is substituted by a    group R² of the formula -L-R³, wherein L is an optional linker group    and R³ is an aromatic hydrocarbyl group.-   2. A compound according to paragraph 1 wherein said compound is    capable of inhibiting steroid sulphatase (STS) activity and/or is    capable of acting as a modulator of cell cycling and/or as a    modulator of apoptosis and/or as a modulator of cell growth.-   3. A compound according to paragraph 1 or 2 of Formula I

-   4. A compound according to paragraph 1 or 2 of Formula II

-   5. A compound according to paragraph 1 or 2 of Formula III

-   6. A compound according to paragraph 1 or 2 of Formula IV

-   7. A compound according to any one of the preceding paragraphs    wherein the A ring of the steroidal ring system is substituted with    a group R⁴, wherein R⁴ is a hydrocarbyl group.-   8. A compound according to paragraph 7 wherein R⁴ is an    oxyhydrocarbyl group.-   9. A compound according to paragraph 8 wherein R⁴ is an alkoxy    group.-   10. A compound according to paragraph 9 wherein R⁴ is methoxy.-   11. A compound according to paragraph 7 wherein R⁴ is an hydrocarbon    group.-   12 A compound according to paragraph 11 wherein R⁴ is an alkyl    group.-   13. A compound according to paragraph 12 wherein R⁴ is ethyl.-   14. A compound according to any one of paragraphs 7 to 13 wherein    the A ring is substituted with R¹ and R⁴ is ortho substituted with    respect to R¹.-   15. A compound according to any one of paragraphs 7 to 14 wherein R⁴    is at position 2 of the A ring.-   16. A compound according to any one of the preceding paragraphs    wherein the compound comprises at least two or more of sulphamate    group, a phosphonate group, a thiophosphonate group, a sulphonate    group or a sulphonamide group.-   17. A compound according to any one of the preceding paragraphs    wherein the compound comprises at least two sulphamate groups.-   18. A compound according to any one of the preceding paragraphs    wherein the compound comprises at least two sulphamate groups,    wherein said sulphamate groups are not on the same ring.-   19. A compound according to any one of the preceding paragraphs    wherein the A ring comprises at least one sulphamate group and    wherein the D ring comprises at least one sulphamate group.-   20. A compound according to any one of the preceding paragraphs    wherein R¹ is a sulphamate group.-   21. A compound according to any one of the preceding paragraphs    wherein R³ is or comprises an aromatic ring.-   22. A compound according to any one of the preceding paragraphs    wherein R³ is or comprises a six membered aromatic ring.-   23. A compound according to any one of the preceding paragraphs    wherein R³ is or comprises a six membered aromatic ring containing    carbon and optionally nitrogen.-   24. A compound according to any one of paragraphs 21 to 23 the    aromatic ring is substituted with a group R⁵.-   25. A compound according to paragraph 24 wherein R⁵ is selected from    hydrocarbyl and halogens.-   26. A compound according to paragraph 24 wherein R⁵ is selected from    hydrocarbon and halogens.-   27. A compound according to paragraph 24 wherein R⁵ is selected from    alkyl and halogens.-   28. A compound according to paragraph 24 wherein R⁵ is selected from    C₁₋₁₀ alkyl (preferably C₁₋₅ alkyl), and halogens.-   29. A compound according to any one of the preceding paragraphs    wherein L is a hydrocarbyl group.-   30. A compound according to any one of the preceding paragraphs    wherein L is a hydrocarbon group.-   31. A compound according to any one of the preceding paragraphs    wherein L is an alkyl group.-   32. A compound according to any one of the preceding paragraphs    wherein L is a C₁₋₁₀ alkyl, preferably C₁₋₅ alkyl, preferably C₁ or    C₂ alkyl.-   33. A compound according to any one of the preceding paragraphs    wherein group R² is selected from

-   34. A compound according to any one of the preceding paragraphs    wherein group R² is in an α conformation.-   35. A compound according to any one of the preceding paragraphs    wherein group R² is in an α conformation on the 17 position of the D    ring.-   36. A method comprising (a) performing a steroid sulphatase assay    with one or more candidate compounds having the formula as defined    in any one of the preceding paragraphs; (b) determining whether one    or more of said candidate compounds is/are capable of modulating STS    activity and/or cell cycling and/or cell growth and/or apoptosis;    and (c) selecting one or more of said candidate compounds that    is/are capable of modulating STS activity and/or cell cycling and/or    cell growth and/or apoptosis.-   37. A method comprising (a) performing a steroid sulphatase assay    with one or more candidate compounds having the formula as defined    in any one paragraphs 1 to 35; (b) determining whether one or more    of said candidate compounds is/are capable of inhibiting STS    activity; and (c) selecting one or more of said candidate compounds    that is/are capable of inhibiting STS activity and/or cell cycling    and/or cell growth and/or apoptosis.-   38. A compound identified by the method according to paragraph 36 or    paragraph 37.-   39. A compound according to any one of paragraphs 1 to 35 for use in    medicine.-   40. A pharmaceutical composition comprising the compound according    to any one paragraphs 1 to 35 optionally admixed with a    pharmaceutically acceptable carrier, diluent, excipient or adjuvant.-   41. Use of a compound according to any one of paragraphs 1 to 35 in    the manufacture of a medicament for use in the therapy of a    condition or disease associated with STS and/or cell cycling and/or    apoptosis and/or cell growth.-   42. Use of a compound according to any one paragraphs 1 to 35 in the    manufacture of a medicament for use in the therapy of a condition or    disease associated with adverse STS levels and/or cell cycling    and/or apoptosis and/or cell growth.

All publications and patents and patent applications mentioned in theabove specification are herein incorporated by reference.

Various modifications and variations of the present invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in chemistry, biology or related fields are intended to bewithin the scope of the following claims.

1. A compound of Formula IV

wherein R¹ is selected from any one of a sulphamate group, a phosphonategroup, a thiophosphonate group, a sulphonate group or a sulphonamidegroup; wherein group R² is of the formula L-R^(3,) wherein L is anoptional linker group and R³ is an aromatic hydrocarbyl group, andwherein the A ring of the steroidal ring system is substituted with agroup R⁴, wherein R⁴ is a hydrocarbyl group.
 2. A compound according toclaim 1 wherein R⁴ is an oxyhydrocarbyl group.
 3. A compound accordingto claim 2 wherein R⁴ is an alkoxy group.
 4. A compound according toclaim 3 wherein R⁴ is methoxy.
 5. A compound according to claim 1wherein R⁴ is an hydrocarbon group.
 6. A compound according to claim 5wherein R⁴ is an alkyl group.
 7. A compound according to claim 6 whereinR⁴ is ethyl.
 8. A compound according to claim 1 wherein the A ring issubstituted with R¹ and R⁴ is ortho substituted with respect to R¹.
 9. Acompound according to claim 1 wherein R⁴ is at position 2 of the A ring.10. A compound according to claim 1 wherein R¹ is a sulphamate group.11. A compound according to claim 1 wherein R³ is or comprises anaromatic ring.
 12. A compound according to claim 11 wherein R³is orcomprises a six membered aromatic ring.
 13. A compound according toclaim 12 wherein R³is or comprises a six membered aromatic ringcontaining carbon and optionally nitrogen.
 14. A compound according toclaim 12 wherein the aromatic ring is substituted with a group R⁵wherein R⁵ is selected from hydrocarbyl and halogens.
 15. A compoundaccording to claim 14 wherein R⁵ is selected from hydrocarbon andhalogens.
 16. A compound according to claim 14 wherein R⁵ is selectedfrom alkyl and halogens.
 17. A compound according to claim 14 wherein R⁵is selected from C₁₋₁₀ alkyl, and halogens.
 18. A compound according toclaim 17, wherein R⁵ is C₁₋₅ alkyl.
 19. A compound according to claim 1wherein L is a hydrocarbyl group.
 20. A compound according to claim 1wherein L is a hydrocarbon group.
 21. A compound according to claim 1wherein L is an alkyl group.
 22. A compound according to claim 1 whereinL is a C₁₋₁₀ alkyl.
 23. A compound according to claim 22 wherein L is aC₁₋₅ alkyl.
 24. compound according to claim 22 wherein L is a C₁ or C₂alkyl.
 25. A compound according to claim 1 wherein group R²is selectedfrom


26. A compound according to claim 1 wherein group R² is in an αconformation.
 27. A pharmaceutical composition comprising the compoundaccording to claim 1 optionally admixed with a pharmaceuticallyacceptable carrier, diluent, excipient or adjuvant.